Full text of "USPTO Patents Application 09869475"

See other formats

WORLD INTEUXCTUAL PROPERTY ORGANIZATION

International Bureac

PCX

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent ClassiCcatlon ^ :

A61K 38A9, 38/18, 4m0, A61P 25/02

(11) InternaUonal PubUcatlon Number: WO 00/62798

(43) International PubUcation Date: 26 October 2000 (26.10.00)

(21) Internaaonal Application Number: PCT/US00/(^765

(22) International Filing Date: 1 1 April 2000 (1 1 .04.00)

(30) Priority Data:
60/129,768

15 April 1999 (15.04.99)

(71) Applicant: ST. ELIZABETH'S MEDICAL CENTER. INC.

[US/US]; 736 Cambridge Street, Boston, MA 02135 (US).

(72) Inventor: ISNER, Jeffrey. M.; 34 Brenton Road, Weston, MA

02193 (US).

(74) Agent: PLUMER, Elizabeth, R.; Wolf. Greenfield & Sacks.
P.C., 600 Atlantic Avenue. Boston. MA 02210 (US).

(81) Designated States: AU. CA. JP. European patent (AT, BE,
CH, CY, DE, DK. ES. FI. PR. GB. GR, IE. IT. LU, MC,
NL, PT. SE).

Published

Without international search report and to be republished
upon receipt of that report.

(54) TiUe: ANGIOGENIC GROWTH FACTORS FOR TREATMENT OF PERIPHERAL NEUROPATHY
(57) Abstract

A me^CKt for treating peripheral neuropathy, particularly ischemic {^ripheral neuropathy, is provided. The method involves
administering to subjects in need of such treatment an effective amount of an angiogenic growth factor to alleviate a symptom of the
neuropathy.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCX on the front pages of pamphlets publishing international applications under the PCX.

Albania

£S

Spain

Lesotho

Slovenia

Annenia

Finland

Lithuania

Slovakia

Austria

France

Luxembourg

Senegal

Australia

Gabon

Latvia

Swaziland

Azerbaijan

United Kingdom

Monaco

Chad

Bosnia and Herzegovina

Georgia

Republic of Moldova

Togo

Barbados

Ghana

Mad^ascar

Tajikistan

Belgium

Guinea

The fonncT Yugoslav

'nnkmenistan

Burkina Faso

Greece

Republic of Macedonia

Tmkcy

Bulgaria

Hungary

Mali

Trinidad and Tobago

Benin

Ireland

Mongolia

Ukraine

Brazil

Israel

Mauritania

Uganda

Belaius

Iceland

Malawi

United Stales of America

Canada

Italy

Mexico

Uzbekistan

Central African Republic

Japan

Niger

Viet Nam

Congo

Kenya

Netherlands

Yugoslavia

Switzerbtnd

Kyrgyzstan

Norway

Zimbabwe

Cdic d'lvone

Democratic People*s

New Zealand

Cameroon

Republic of Korea

Poland

China

Republic of Korea

Portugal

Cuba

Kazakstan

Romania

Ct^ch Republic

Saim Luda

Rosstan Fbderaiion

Germany

Liechtenstein

Sudan

Denmark

Sri Lanka

Sweden

Estonia

Liberia

Smgapore

wo 00/62798 PCT/USOO/09765

-1-

ANGIOGENIC GROWTH FACTORS
FOR TREATMENT OF PERIPHERAL NEUROPATHY
Field of the Invention

This invention relates to methods and compositions for the treatment of peripheral
5 neuropathy, such as ischemic peripheral neuropathy. The methods involve administering
an angiogenic growth factor to alleviate a symptom of a peripheral neuropathy and,
optionally, enhance nerve regeneration in a mammal.

Background of the Invention
Peripheral neuropathy generally refers to a disorder that affects the peripheral

10 nerves, most often manifested as one or a combination of motor, sensory, sensorimotor,
or autonomic neural dysfimction. Peripheral neuropathies can be genetically acquired,
can be induced by a toxic agent, or can result from peripheral ischemia or from a
systemic disease. Genetically-acquired peripheral neuropathies include Refsum's
disease, Abetalipoproteinemia, Tangier disease, Krabbe's disease. Metachromatic

15 leukodystrophy, Fabry's disease, Dejerine-Sottas syndrome, Charcot-Marie Tooth
Disease (also known as Peroneal Muscular Atrophy, or Hereditary Motor Sensory
Neuropathy) and others. Exemplary toxic agents which cause neurotoxicities include
therapeutic drugs such as antineoplastic agents, contaminants in foods, and environmental
and mdustrial pollutants. Ischemic peripheral neuropathies include, but are not limited to,

20 diabetic polyneuropathy. Peripheral neuropathies associated with a systemic condition
include uremia and alcoholic polyneuropathy among other disorders.

Ischemic peripheral neuropathy, particularly when it develops in the absence of
diabetes, has received limited study, despite the fact that it may be a prominent feature of
patients with peripheral vascular disease. Among diabetics, peripheral neuropathy is

25 common and ultimately accounts for significant morbidity. Typically, symptoms are
dominated by sensory defects. (Tomlinson DR, et al. Diabetes 1997;46:S43-S49). The
ultimate consequence of such sensory deficits involving the lower extremities may be
foot ulceration initiated by traumatic injury that is inapparent to the patient. Indeed, it has
been reported that 20% of all hospital admissions among diabetic patients in the United

30 States are for foot problems. (Reiber GE, et al., in Harris MI, et al, (eds): Diabetes in

America. Washington, National Institute of Diabetes and Digestive and Kidney Diseases,

wo 00/62798

-2-

PCT/USOO/09765

1995, pp 409-427). That such ulcerations may lead to lower extremity amputation
(Parkhouse N, et al,,NEnglJMed 1988;31 8:1306-1309) is borne out by the fact that the
rate of lower limb amputation is fifteen times higher in diabetic versus non-diabetic
patients (Veves A, et al., Diabetes 1998;47:457-463). Even with intensive insulin
5 therapy, as reported in the Diabetes Control and Complications Trial (DCCT), the

incidence of new clinically detected neuropathy per patient per year was 3.1% in the non-
retinopathy group and 7.0% in the group with baseline retinopathy; with conventional
therapy, the incidence of neuropathy increased to 9.8% and 16.1% with and without
retinopathy (The Diabetes Control and Complications Trial Research Group, N Engl J

10 Med 1993;329:977-988). When loss of sensation is compounded by loss of control over
blood flow due to autonomic neuropathy and lower extremity vascular obstruction, the
threat of limb loss is exacerbated. In the case of peripheral artery disease, hospital
mortality, length of hospitalization, and complications resulting from surgery are all
increased in the presence of diabetes (Currie CJ, et al., Diabetes Care 1998;21:42-48).

15 In view of the foregoing, a need still exists to better understand the molecular

processes underlying peripheral neuropathy, and to develop improved drug therapies to
replace or supplement the existing methods for treating peripheral neuropathies,
particularly ischemic peripheral neuropathy. Preferably, such drug therapies would be
designed to reduce or prevent nerve damage at its earliest stages and to enhance

20 peripheral nerve repair following diagnosis and treatment.

Summary of the Invention
The invention is based, in part, on the observation that a nimiber of patients
receiving angiogenic growth factor treatment for vascular insufficiency appeared to
exhibit an improvement in sensory neuropathy. To test Applicant's hypothesis that the

25 angiogenic grov^ factor either directly or indirectly improved the peripheral neuropathy.
Applicant developed an animal model of hindlimb ischemia which exhibits severe
peripheral neuropathy and which, in contrast to existing in vitro and in vivo methods, is
predictive of an in vivo therapeutic effect of an agent for treating a peripheral neuropathy,
administered (intramuscularly) to this animal model a vector containing a nucleic acid

30 encoding VEGF ("VEGF vector"), and discovered that this angiogenic growth factor

attenuated the development of ischemic peripheral neuropathy and enhanced the recovery
of established ischemic peripheral neuropathy. Thus, Applicant describes herein newly

wo 00/62798

-3-

PCTA;S00/09765

discovered functions for VEGF and other angiogenic growth factors, namely, the ability
to prevent or reduce ischemia induced nerve damage at its earliest stages and the ability
to enhance peripheral nerve repair following the onset of peripheral neuropathy.
Accordingly, the instant invention is directed to compositions and methods that are based
5 upon the discovery of these newly-discovered functions for angiogenic growth factors.
Exemplary conditions that are characterized by peripheral neuropathy include: (a) an
ischemic peripheral neuropathy; (b) a neuropathy associated with a systemic condition;
(c) a toxin-induced peripheral neuropathy; and (d) a genetically acquired peripheral
neuropathy.

10 According to one aspect of the invention, a method for treating a condition

characterized by peripheral neuropathy in a subject is provided. In certeiin preferred
embodiments, the subject is otherwise free of symptoms calling for treatment with an
angiogenic growth factor. The method involves administering at least one angiogenic
growth factor (an "angiogenic growth factor nucleic acid" or an "angiogenic growth

15 factor polypeptide") to a subject in need of such treatment in an amount effective to

alleviate a symptom of peripheral neuropathy in the subject. The symptom of peripheral
neuropathy can be one or more of the symptoms which are used by the skilled medial
professional to diagnose a peripheral neuropathy.

Exemplary angiogenic growth factors (including all genes and isoforms of each

20 gene product) for use in accordance with the methods of the invention include: vascular
endothelial cell growth factor (VEGF), acidic fibroblast growth factor (aFGF), basic
fibroblast growth factor (bFGF), epidermal growth factor, transforming growth factors a
and P, platelet-derived endothelial growth factor, platelet-derived growth factor, tumor
necrosis factor a, hepatocyte growth factor (scatter factor), erythropoietin, colony

25 stimulating factor (CSF), macrophage-CSF(M-CSF), granulocyte/macrophage CSF (GM-
CSF), angiopoietin 1 and 2, and nitric oxide synthase (NOS). The nucleic acid and amino
acid sequences for these and other angiogenic growth factors are available in public
databases such as GenBank and in the literature. The preferred angiogenic growth factor
is VEGF, more preferably, a VEGF nucleic acid. In certain particularly preferred

30 embodiments, the VEGF nucleic acid is administered to the subject in conjunction ynth a
second angiogenic growth factor nucleic acid which, preferably, is bFGF. The
compositions and methods of the invention are useful for replacing existing drug

wo 00/62798

-4-

PCTAJSQO/09765

therapies, as well as for improving the effectiveness of existing therapies for treating
conditions that are characterized by peripheral neuropathy. In general, such conditions
are diagnosed by detecting one or a combination of motor, sensory, sensorimotor, or
autonomic neural dysfunctions in the subject.

5 An "angiogenic growth factor" embraces an "angiogenic growth factor nucleic

acid" and an "angiogenic growth factor polypeptide". As used herein, an "angiogenic
growth factor polypeptide" refers to any protein, polypeptide, mutein or portion thereof
that is capable of inducing endothelial cell growth. VEGF is a particularly preferred
angiogenic growth factor (e.g., VEGF 1 (also referred to as VEGF A); VEGF 2 (also

10 referred to as VEGF C); VEGF B; and VEGF D). An angiogenic growth factor nucleic
acid refers to a nucleic acid which encodes an angiogenic growth factor polypeptide. The
invention embraces the administration of angiogenic growth factor nucleic acids and
polypeptides for the treatment of peripheral neuropathies. The invention also embraces
agents that upregulate expression of an angiogenic growth factor polypeptide in vivo.

15 The complete coding sequence for representative preferred human angiogenic

growth factors cDNA and predicted amino acid sequence are available in public
databases such as GenBank and in literature. In particular, certain of the VEGF genes,
isoforms, fragments, and analogs thereof that are useful for practicing the claimed
invention are described in GenBank Accession Nos. NM 003376 ("Homo sapiens

20 vascular endothelial growth factor (VEGF) mRNA"); NM 003377 ("Homo sapiens
vascular endothelial growth factor B (VEGFB) mRNA"); NM 005429 ("Homo sapiens
vascular endothelial growth factor C (VEGFC) mRNA"); NM 004469 ("Homo sapiens c-
fos induced growth factor (vascular endothelial growth factor D) (FIGF) mRNA); AF
024710 ("Homo sapiens vascular growth factor (VEGF (165)) mRNA, 3'UTR, mRNA

25 sequence"); and U.S. Patent Nos. 6,013,780 ("VEGF145 expression vectors"); 5,935,820
("Polynucleotides encoding vasctdar endodielial growth factor 2"); 5,607,918 ("Vascular
endothelial growth factor-B and DNA coding therefor"); and 5,219,739 ("DNA
sequences encoding bVEGF 120 and hVEGF 121 and methods for the production of
bovine and human vascular endothelial cell growth factors, bVEGFi2o and hVEGF^i"),

30 including references cited therein, the entire contents of the foregoing accession numbers,
patent docimients, and references are incorporated in their entirety by reference.

wo 00/62798

PCT/USOO/09765

-5-

The preferred angiogenic growth factor nucleic acids of the invention encode the
above-identified angiogenic growth factor polypeptides, as well as their homologs and
alleles and functionally equivalent fi-agments or variants of the foregoing. For example,
human VEGF 1 (VEGF A) exists in four principal isoforms, phVEGFi2i; phVEGF]45;
5 phVEGFi65; and phVEGFigg. Preferably, the angiogenic growth factor nucleic acid has
the nucleotide sequence encoding an intact human angiogenic growth factor polypeptide,
i.e., the complete coding sequence of the gene encoding a human angiogenic grovs^
factor; however the invention also embraces the use of nucleic acids encoding fi-agments
of an intact angiogenic growth factor.

10 In general, the angiogenic growth factor nucleic acid is operatively coupled to a

promoter that can express the angiogenic growth factor in a targeted cell (e.g., an
endothelial cell, a nerve cell, a muscle cell). Optionally, the nucleic acid is contained in
an appropriate expression vector (e.g., plasmid, adenoviral vector, modified adenoviral
vector, retroviral vector, liposome) to more efficiently genetically modify the targeted

15 cell and achieve expression of the angiogenic growth factor.

According to another aspect of the invention, an alternative method for treating a
condition characterized by peripheral nexiropathy in a subject is provided. The method
involves: administering at least one angiogenic growth factor to a subject in need of such
treatment in an amount and for a period of time effective to alleviate a symptom of

20 peripheral neuropathy in the subject. The symptom of peripheral neuropathy can be one
or more of the symptoms which are used by the skilled medical professional to diagnose a
peripheral neuropathy. The period of time that is effective for alleviating a symptom of
peripheral neuropathy is significantly greater than the period of time during which an
angiogenic growth factor typically is administered to a subject for the purpose of

25 revascularization in an ischemic tissue. In general, angiogenic growth factors are
administered to a patient for a period of up to, and including, about twelve weeks to
enhance blood vessel development in ischemic tissue. In contrast, in the preferred
embodiments of this aspect of the invention, administration of the angiogenic growth
factor is for greater than twelve weeks; more preferably, greater than eighteen weeks; and

30 most preferably, greater than about twenty-four weeks. In some instances, treatment for
the pmposes of this aspect of the invention is continued for at least six months to several

wo 00/62798

PCT/USOO/09765

-6-

years, and more preferably, from six months to one, two, three years or for the patient's
lifetime in the case of chronic peripheral neuropathy.

According to yet another aspect of the invention, a further method for treating a
condition characterized by peripheral neuropathy in a subject is provided. The method
5 involves administering at least one angiogenic growth factor to a subject in need of such
treatment in an amount effective to alleviate a symptom of peripheral neuropathy in the
subject, wherein administering is by intramuscular injection into a tissue at an injection
site that is proximate to a nerve which is suspected of a neuropathy and that is distal to an
injection site that would be selected for the puqDOse of revascularization (e.g.,

10 neovascularization). In general, intramuscular injection into a tissue for the purpose of
revascularization in ischemic tissue is accomplished by localized delivery of the
angiogenic growth factor to the site of a vascular blockage. In contrast, according to the
embodiments in which a subject also presents with a vascular insufficiency, intramuscular
injection of angiogenic growth factors, preferably, is into the tissue at a location which

15 excludes these locations. The preferred locations into which the angiogenic factors are
intramuscularly injected for the purpose of treating peripheral neuropathy include sites
which are proximate to a nerve which is suspected of a neuropathy. An amount of the
angiogenic growth factor is administered to alleviate a symptom of a peripheral
neuropathy. The symptom of peripheral neuropathy can be one or more symptoms which

20 are used by the skilled medical professional to diagnose a peripheral neuropathy.

According to yet another aspect of the invention, a method for treating a subject
who has sustained a peripheral nerve injury is provided. The method involves
administering at least one angiogenic growth factor to a subject in need of such treatment
in an amount effective to enhance peripheral nerve regeneration. Preferably, the subject

25 is otherwise free of symptoms calling for treatment vwth an angiogenic growth factor.
The angiogenic growth factor nucleic acids and polypeptides and exemplary conditions
which are characterized by peripheral neuropathy are as described above.

It is to be understood that an angiogenic growth factor polypeptide can be used in
place of an angiogenic growth factor nucleic acid in treating any of the foregoing

30 conditions. Thus, according to still another aspect of the invention, pharmaceutical
preparations are provided that contain an angiogenic growth factor nucleic acid or an
angiogenic growth factor polypeptide. The pharmaceutical preparations contain the

wo 00/62798

PCT/USOO/09765

-7-

above-described angiogenic growth factors, together with a pharmaceutically-acceptable
carrier. Preferably, the angiogenic growth factors are present in the compositions in an
amount effective for treating a peripheral neuropathy. The angiogenic growth factors are
particularly useful for the treatment of ischemic peripheral neuropathy. Preferably, this
5 amoimt is sufficient to enhance nerve regeneration in vivo.

According to still another aspect of the invention, the above angiogenic growth
factors (angiogenic growth factor nucleic acids and angiogenic growth factor
polypeptides), alone or in combination, are used in the preparation of medicaments for
the treatment of a peripheral neuropathy. The method involves placing the angiogenic

10 growth factor(s) in a pharmaceutically-acceptable carrier. The preferred angiogenic
growth factors are as described above.

It is noteworthy that in certain embodiments, the preferred subjects treated
according to the methods set forth above are otherwise free of symptoms calling for
angiogenic growth factor treatment, either by administration of the angiogenic growth

15 factor polypeptide or by an angiogenic growth factor nucleic acid. Thus, in certain select
embodiments, the subjects are not otherwise being treated using a gene therapy protocol
or, if being treated using gene therapy, the protocol for the methods of the invention
differ in the dosage or duration of treatment (greater than about twelve weeks) and/or the
site of intramuscular injection (in the proximity of a nerve suspected of a neuropathy).

20 The invention also contemplates the use of angiogenic growth factors in

experimental model systems to determine the role that angiogenic growth factors play in
the repair of peripheral nerves or in mediating an adverse health consequence occurring
as a result of peripheral neuropathy. An ischemic animal model of peripheral neuropathy
is described in the Examples and can be used to select agents for treatment of this

25 condition. The agent (e.g., an angiogenic growth factor as described above) is
administered to the animal, locally or systemically, and the animal's response is
monitored and compared to control animals that do not receive the angiogenic growth
factors. In this manner, additional agents which are useful for treating peripheral
neuropathies can be identified.

30 These and other aspects of the invention will be described in greater detail below.

Throughout this disclosure, all technical and scientific terms have the same meaning as

wo 00/62798 PCT/USOO/09765

-8-

commonly understood by one of ordinary skill in the art to which this invention pertains
unless defined otherwise.

Detailed Description of the Drawings

Figure lA shows the motor nerve conduction velocity for non-diabetic and
5 diabetic animals which received saline or VEGF; and

Figure IB shov^ sensory nerve conduction velocity for non-diabetic and diabetic
animals which received saline and VEGF.

This application, particularly the Examples, may refer to Figures; however, none
of the figures are essential for enablement of the invention.
10 Detailed Description of the Invention

The invention is based, in part, on the observation that a number of patients
receiving an angiogenic growth factor to treat a vascular insufficiency appeared to exhibit
an improvement in sensory neuropathy. Accordingly, Applicant hypothesized that the
angiogenic growth factor either directly or indirectly improved the peripheral neuropathy.
15 To test this hypothesis. Applicant developed an animal model of hindlimb ischemia
which exhibits severe peripheral neuropathy and which resembles and is predictive of a
human peripheral neuropathy. Applicant then administered (intramuscularly) to the
animal model, a vector containing a nucleic acid encoding VEGF ("VEGF vector"), and
discovered that this angiogenic growth factor attenuated the development of ischemic
20 peripheral neuropathy and enhanced the recovery of established ischemic peripheral
neuropathy. Based on these discoveries. Applicant describes herein new functions for
VEGF and other angiogenic growth factors, namely, the ability to prevent or reduce
ischemia induced nerve damage at its earliest stages as well as the ability to enhance
peripheral nerve repair. Accordingly, the instant invention is directed to compositions
25 and methods that are based upon the discovery of these newly-discovered fiinctions for
angiogenic growth factors.

As used herein, "peripheral neuropathy" refers to a disorder affecting a segment
of the peripheral nervous system. The invention involves using an angiogenic growth
factor to reduce a neuropathology including, but not limited to, a distal sensorimotor
30 neuropathy, or an autonomic neuropathy such as reduced motility of the gastrointestinal
tract or atony of the urinary bladder. Preferred neuropathies that can be treated with the
angiogenic growth factors of the invention also include neuropathies associated with

wo 00/62798

PCT/USOO/09765

-9-

ischemic disease, neuropathies associated with a systemic disease, e.g., post-polio
syndrome; genetically acquired neuropathies, e.g., Charcot-Marie-Tooth disease; and
neuropathies caused by a toxic agent, e.g., a chemotherapeutic agent, such as vincristine.
Each of these categories of conditions is discussed in more detail below.
5 According to one aspect of the invention, a method for treating a condition

characterized by peripheral neuropathy in a subject is provided. The method involves
administering at least one angiogenic growth factor (an "angiogenic growth factor nucleic
acid" or an "angiogenic growth factor polypeptide") to a subject in need of such treatment
in an amount effective to alleviate a symptom of peripheral neuropathy in the subject.

10 The symptom of peripheral neuropathy can be one or more of the symptoms which are
used by the skilled medical professional to diagnose a peripheral neuropathy. Exemplary
angiogenic growth factors for use in accordance with the methods of the invention are
described below. The nucleic acid and amino acid sequences for these and other
angiogenic growth factors are available in public databases such as GenBank and in the

15 literature. Preferably, the subject is otherwise free of symptoms calling for treatment
with an angiogenic growth factor.

According to another aspect of the invention, an alternative method for treating a
condition characterized by peripheral neuropathy in a subject is provided. The method
involves: administering at least one angiogenic growth factor to a subject in need of such

20 treatment in an amount and for a period of time effective to alleviate a symptom of

peripheral neuropathy in the subject. The symptom of peripheral neuropathy can be one
or more of the symptoms which are used by the skilled medical professional to diagnose a
peripheral neuropathy. The period of time that is effective for alleviating a symptom of
peripheral neuropathy is significantly greater than the period of time during which an

25 angiogenic growth factor is administered to a subject for the purpose of revascularization
of an ischemic tissue. Typically, angiogenic growth factors are administered to a patient
for a period of up to, and including, about twelve weeks for enhancing blood vessel
development in ischemic vascular tissue. In contrast, in the preferred embodiments of
this aspect of the invention, administration of the angiogenic growth factor is for greater

30 than about twelve weeks; more preferably, greater than eighteen weeks; and most

preferably, greater than about twenty-four weeks. In some instances, treatment for the
purposes of this aspect of the invention is continued for at least six months to several

wo 00/62798 PCT/USOO/09765

-10-

years, and more preferably, from six months to one, two, three years, or for the patient's
lifetime in the case of chronic peripheral neuropathy. Repeated injections using
controlled release microparticles containing the angiogenic growth factors can be used for
this purpose.

5 According to yet another aspect of the invention, a further method for treating a

condition characterized by peripheral neuropathy in a subject is provided. The method
involves administering at least one angiogenic grov^ factor to a subject in need of such
treatment in an amount effective to alleviate a symptom of peripheral neuropathy in the
subject, wherein administering is by intramuscular injection into a tissue at an injection

10 site that is proximate to a nerve which is suspected of a neuropathy and that is distal to an
injection site that would be selected for the purpose of revascularization (e.g.,
neovascularization) in an ischemic tissue. In general, intramuscular injection into a tissue
for the purpose of revascularization in ischemic tissue is accomplished by localized
delivery of the angiogenic growth factor to the site of a vascular blockage. In contrast,

15 according to the embodiments in which a subject also presents v^th a vascular

insufficiency, intramuscular injection of angiogenic grov^ factors, preferably, is into a
tissue at a location which excludes these locations. The locations into which the
angiogenic factors are intramuscularly injected for the purpose of treating peripheral
neuropathy include muscle which is proximate to a nerve which is suspected of a

20 neuropathy.

25 is otherwise free of symptoms calling for treatment v^th an angiogenic growth factor.

As used herein, an "angiogenic growth factor" embraces an "angiogenic growth
factor nucleic acid" and an "angiogenic growth factor polypeptide". An "angiogenic
grov^ factor polypeptide" refers to any protein, polypeptide, mutein or portion that is
capable of inducing endothelial cell grov^. VEGF is a particularly preferred angiogenic

30 growth factor. An angiogenic growth factor nucleic acid refers to a nucleic acid which
encodes an angiogenic growth factor. The invention embraces the administration of
nucleic acids or polypeptides for the treatment of peripheral neuropathies.

wo 00/62798

PCT/USOO/09765

-11-

Homologs and alleles of the nucleic acid and amino acid sequences reported for
the angiogenic growth factors, such as those identified herein, also are embraced within
the definition of an "angiogenic growth factor". In addition, the angiogenic growth
factor nucleic acids of the invention include nucleic acids which code for the angiogenic
5 growth factor polypeptides having the sequences reported in the public databases and/or
literature, but which differ fi-om the naturally occurring nucleic acid sequences in codon
sequence due to the degeneracy of the genetic code. The invention also embraces
isolated functionally equivalent fragments, variants, and analogs of the foregoing nucleic
acids; proteins and peptides coded for by any of the foregoing nucleic acids; and

10 complements of the foregoing nucleic acids. Particularly preferred fragments of the
VEGF nucleic acid and VEGF polypeptides are identified below.

The angiogenic growth factor nucleic acid may be any nucleic acid (DNA or
RNA) including genomic DNA, cDNA and mRNA, encoding an angiogenic growth
factor which can be used to express a growth factor, e.g., a protein, polypeptide, mutein

15 or portion thereof, that is capable of inducing either directly or indirectly, the formation
of new blood vessels (Folkman, et al,, Science, 235:442-447 (1 987)). These include, for
example, vascular endothelial growth factor (VEGF), acidic fibroblast growth factor
(aFGF) (Bjomsson, et al., Proc. Natl. Acad. Sci. USA, 88:8651-8655, (1991)), basic
fibroblast growth factor (bFGF) (Schwarz, et al., J. Vase Surg., 5:280-288, (1987)),

20 epidermal growth factor (EGF), transforming grov^h factors a and P (TGF-a and TGF-P),
platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor
(PDGF) itself, tumor necrosis factor a (TNF-a), hepatocyte growth factor (HGF),
erythropoietin, colony stimulating factor (CSF), macrophage-CSF(M-CSF),
granulocyte/macrophage CSF (GM-CSF) and nitric oxide synthase (NOS). See,

25 Klagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991); Folkman, et al., J. Biol.
Chem., 267:10931-10934 (1992) and Symes, et al.. Current Opinion in Lipidology,
5:305-312 (1994). Muteins or fragments of an angiogenic growth factor may be used
provided they induce nerve regeneration or alleviate a symptom of peripheral neuropathy.
The feasibility of using recombinant formulations of angiogenic growth factors to

30 expedite and/or augment collateral artery development in animal models of myocardial
and hindlimb ischemia has been reported. See, Baffour, et al., supra (bFGF); Pu, et al..
Circulation, 88:208-215 (1993) (aFGF); Yanagisawa-Miwa, et al., supra (bFGF); Ferrara,

wo 00/62798

PCTAJS00/0976S

-12-

et al., Biochem. Biophys. Res. Commun., 161:851-855 (1989) (VEGF). In addition,
therapeutic angiogenesis has been reported in the same or closely related models
following administration of recombinant endothelial cell growth factor (ECGF) (Pu, et.
al.. Circulation, 88:208-215 (1993)) and VEGF (Takeshita, et al.. Circulation, 90:228-
5 234 (1994) supra). Previous studies, employing the animal model of chronic limb
ischemia, reported an efficacy of intra-muscular endothelial cell growth factor (ECGF)
(Pu, et al.. Circulation, 88:208-215 (1993) or VEGF (Takeshita, et al., Circulation,
90:228-234 (1994) supra) administration. None of these references have suggested a role
for angiogenic growth factors for treating peripheral neuropathy.

10 VEGF is a particularly preferred angiogenic growth factor. Any of the VEGF

isoforms (e.g., VEGF 1, 2, 3, 4, and 5) may be used in accordance v^th the methods of
the invention. VEGF reportedly is an endothelial cell-specific mitogen (Ferrara, et al.,
Biochem Biophys Res Commun., 161 :85l-855, (1989), Keck, et al.. Science,
246:1309-1312 (1989), and Plouet, et al., Embo J., 3801-3806 (1989)). VEGF was

15 purified independently as a tumor-secreted factor that included vascular permeability by
the Miles assay (Keck, et al., supra, and Connolly, et al., J. Biol. Chem.,
264:20017-20024 (1989)), and thus has an altemate designation, vascular permeability
factor (VPF). Two features distinguish VEGF fi-om other heparin-binding, angiogenic
growth factors. First, the NH2 terminus of VEGF is preceded by a typical signal

20 sequence; therefore, unlike bFGF, VEGF can be secreted by intact cells. Second, its
high-affinity binding sites, shown to include the tyrosine kinase receptors Flt-1 and
Flt-1/KDR are present on endothelial cells. Ferrara, et al., supra, and Conn, et al., Proc.
Natl. Acad. Sci. USA, 87:1323-1327 (1990). DNA encoding VEGF is disclosed in U.S.
Pat. No. 5,332,671, the disclosure of which is herein incorporated by reference.

25 Preferably, the angiogenic growth factor contains a secretory signal sequence that

facilitates secretion of the protein. Angiogenic growth factors having native signal
sequences, e.g., VEGF, are preferred. Angiogenic growth factors that do not have native
signal sequences, e.g., bFGF, can be modified to contain such sequences using routine
genetic manipulation techniques. See, Nabel, et al.. Nature, 362:844 (1993).

30 The nucleotide sequence of numerous peptides and proteins, including the

angiogenic growth factors, are readily available through a number of computer data
bases, for example, GenBank, EMBL and Swiss-Prot. Using this information, a DNA or

wo 00/62798 PCT/USOO/09765

-13-

RNA segment encoding the desired may be chemically synthesized or, alternatively, such
a DNA or RNA segment may be obtained using routine procedures in the art, e.g, PGR
amplification.

To simplify the manipulation and handling of the nucleic acid encoding the
5 growth factor, the nucleic acid preferably is inserted into a cassette where it is operably
linked to a promoter. The promoter must be capable of driving expression of the mitogen
in the desired target host cell, e.g., an endothelial cell, a muscle cell, a nerve cell. The
selection of appropriate promoters can readily be accomplished. Preferably, a high
expression promoter is used. An example of a suitable promoter is the 763 -base-pair

10 cytomegalovirus (CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et al.. Hum
Gene Ther 4:151 (1 993)) and MMT promoters may also be used. Certain proteins can be
expressed using their native promoter. Other elements that can enhance expression can
also be included such as an enhancer or a system that results in high levels of expression
such as a tat gene and tar element. This cassette can then be inserted into a vector, e.g., a

1 5 plasmid vector such as pUCl 1 8, pBR322, or other known plasmid vectors, that includes,
for example, an E. coli origin of replication. See, Sambrook, et al.. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). A plasmid vector may
also include a selectable marker such as the P-lactamase gene for ampicillin resistance,
provided that the marker polypeptide does not adversely effect the metabolism of the

20 organism being treated. The cassette can also be bound to a nucleic acid binding moiety
in a synthetic delivery system, such as the system disclosed in WO 95/122618.

If desired, the DNA may also be used with a micro delivery vehicle such as
cationic liposomes and adenoviral vectors. For a review of the procedures for liposome
preparation, targeting and delivery of contents, see Mannino and Gould-Fogerite, Bio

25 Techniques, 6:682 (1988). See also. Feigner and Holm, Bethesda Res. Lab. Focus, 1 1
(2):21 (1989) and Maurer, R. A., Bethesda Res. Lab. Focus, 1 1(2):25 (1989).

Replication-defective recombinant adenoviral vectors, can be produced in
accordance with known techniques. See, Quantin, et al., Proc. Natl. Acad. Sci. USA,
89:2581-2584 (1992); Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); and

30 Rosenfeld, et al.. Cell, 68:143-155 (1992).

In certEiin situations, it may be desirable to use nucleic acids encoding two or
more different proteins in order to optimize the therapeutic outcome. For example, DNA

wo 00/62798 PCTA;S00/a9765

-14-

encoding two angiogenic growth factors, e.g., VEGF and bFGF, can be used to provide
an improved treatment over the use of bFGF alone. Alternatively, an angiogenic growth
factor nucleic acid can be combined with other genes or their encoded gene products to
enhance the activity of targeted cells, while simultaneously inducing angiogenesis, if
5 desired, including, for example, nitric oxide synthase, L-arginine, fibronectin, urokinase,
plasminogen activator and heparin.

In order to facilitate injection, the nucleic acid is formulated with a
pharmaceutically acceptable carrier. Examples of suitable carriers include, saline,
albxmiin, dextrose and sterile water. The nucleic acid is injected into the ischemic tissue

10 using standard injection techniques by use of, for example, a hypodermic needle.

Hypodermic needle sizes from no. 29 to no. 16 are preferred. The nucleic acid may also
be injected by an externally applied local injection apparatus, such as that used to inject
antigens for allergy testing; or a transcutaneous "patch" capable of delivery to
subcutaneous muscle. In general, the effective dose of the nucleic acid will be a function

15 of the particular expressed protein, the target tissue, the patient and his or her clinical
condition. Effective amounts of DNA typically are between about 1 and 4000 pg, more
preferably from about 1000 to 4000 ^g and, most preferably, from about 2000 to 4000

Once injected, the nucleic acid capable of expressing the desired angiogenic
20 grov^ factor is taken up and expressed by the cells of the tissue. Because the vectors
containing the nucleic acid of interest are not normally incorporated into the genome of
the cells, expression of the protein of interest takes place for only a limited time.
Typically, the angiogenic growth factor is expressed at therapeutic levels for about two
days to several weeks, preferably for about 1-2 weeks. Reinjection of the DNA can be
25 utilized to provide additional periods of expression of the angiogenic growth factor. If
desired, a retrovirus vector can be used to incorporate the heterologous DNA into the
genome of the cells and, thereby, increase the length of time during which the therapeutic
polypeptide is expressed, from several weeks to indefinitely.

The invention is not limited to treatment of ischemic tissue, but rather, is useful
30 for treating peripheral neuropathies of various origin. Exemplary conditions that are

characterized by peripheral neuropathy are known to those of ordinary skill in the art and
include, but are not limited to, the following categories of disorders: (a) ischemic

wo CO/62798 PCTAJSOO/09765

-15-

peripheral neuropathies; (b) toxin-induced peripheral neuropathies; (c) neuropathies
associated with systemic disease; and (d) genetically acquired peripheral neuropathies.

Exemplary ischemic peripheral neuropathies include neuropathies associated with
ischemic tissues such as those associated with a diabetic condition, peripheral vascular
5 disease, or other vascular insufficiency.

Exemplary toxin-induced peripheral neuropathies are described in U.S. Patent No.
5,648,335, entitled "Prevention and treatment of peripheral neuropathy", issued to Lewis,
et al., and include neuropathies that are caused by neurotoxic agents including,
therapeutic drugs, antineoplastic agents, contaminants in foods or medicinals, and

10 environmental and industrial pollutants. By "toxic agent", or neurotoxic agent, is meant
a substance that through its chemical action injures, impairs, or inhibits the activity of a
component of the nervous system. The list of neurotoxic agents that cause neuropathies is
lengthy, and includes, but is not limited to, neoplastic agents such as vincristine,
vinblastine, cisplatin, taxol, or dideoxy-compounds, e.g., dideoxyinosine; alcohol;

15 metals; industrial toxins involved in occupational or environmental exposure;

contaminants of food or medicinals; or over-doses of vitamins or therapeutic drugs, e.g.,
antibiotics such as penicillin or chloramphenicol, or megadoses of vitamins A, D, or B^.
An extensive, although not complete, list of chemical compounds with neurotoxic
side-effects is found in Table 1 . Although this list provides examples of neurotoxic

20 compoimds, it is intended to exemplify, not limit, the scope of the invention. Other toxic
agents can cause neuropathies, and can be characterized by methods known to one skilled
in the art. By "exposure to a toxic agent" is meant that the toxic agent is made available
to, or comes into contact v^th, a mammal of the invention. Exposure to a toxic agent can
occur by direct administration, e.g., by ingestion or administration of a food, medicinal,

25 or therapeutic agent, e.g., a chemotherapeutic agent, by accidental contamination, or by
environmental exposure, e.g., aerial or aqueous exposure.

TABLE 1: AGENTS THAT CAUSE PERIPHERAL NEUROPATHY

AGENT

ACTIVITY

Acetazolamide

diuretic

Acrylimide

flocculent, grouting agent

Adriamycin

antineoplastic

alcohol (ethanol) solvent

recreational drug

wo 00/62798

-16-

PCT/USOO/09765

Almitrine

resniratorv stimulant

Amiodarone

antiarrhythmic

Amnhotericin

antimicrobial

Ar^jPTiic

herbicide insecticide

Aurothioel uco se

antirheumati c

B arbi turates

anticonvulsant sedative

Buckthorn

toxic berry

Carbamates

insecticide

carbon disulfide f CS-^"!

industrial

chloramnhenicol

antibacterial

phlnrnniiirip

antimalarial

CUllililAlCU iCll

f*li n 1 6* <? tvra tn i n e

antih vnerl innnrntf^i nf*m i c

antinennla^tic

rlinniiinnl ampbipide

antibacterial

rnlp<!tinnl

an ti h vnerl i nnnrntpi n pm t c

colchicine

20ut sunnressant

rnli<!tin

antimicrobial

rvcln^prinp

antibacterial

cvtarahine

antineonlastic

dansone

dermatolosic including lenrosv

wWA XAAU-lV/XV/^A W AAA WA U.U.AAAK AWL/X T

dideoxvcvtidine

antineonlastic

UX X U AX W L/ A UiJ VX W

dideoxvinosine

\iXXUWV/V T UXx/iSUxW

antine on 1 asti c

d 1 d en wth vm 1 d i Ti e

antiviral

disulfiram

anti alcohol

WXX^XUAW\/A Av A

dftYnnihif^in

antinpnnla<3tip

CU A Li i 1& V/ LI 1 do Li W

pthamhiitol

wUiCUiiUULVfi

antibartprial

ethionamide

antibacterial

giutethimide

sedative, hypnotic

gold

antirheumatic

hexacarbons solvents

hormonal contraceptives

hexamethylolmelamine

fireproofing, creaseproofing

wo 00/62798

-17-

PCTAJS0O/Oy76S

hydralazine

antihypertensive

hydroxychloroquine

antirheumatic

imipramine

antidepressant

indomethacin

anti-inflammatory

inorganic lead

toxic metal in paint, etc.

isoniazid

antituberculous

lithiiim

antidepressant

methylmercury

industrial waste

metformin

antidiabetic

methylhydrazine

synthetic intermediate

metronidazole

antiprotozoal

misonidazole

radiosensitizer

nitrofurantoin

urinary antiseptic

nitrogen mustard antineoplastic

nerve gas

nitrous oxide

anesthetic

organophosphates

insecticides

ospolot

anticonvulsant

penicillin

antibacterial

perhexiline

antiarrhythmic

perhexiline maleate

antianhythmic

phenytoin

anticonvulsant

platinum

drug component

primidone

anticonvulsant

procarbazine

antineoplastic

pyridoxine

vitamin B6

sodium cvanate

anti-sickling

streptomycin

antimicrobial

sulphonamides

antimicrobial

suramin

antineoplastic

tamoxifen

antineoplastic

taxol

antineoplastic

wo 00/62798

-18-

PCT/USOO/09765

thnltdftiniHe

antileorous

thalliiiTn
uxcuix mil

rat poison

tri a m t eren e

diuretic

trimpfVivltin

U JlllCUIjrlliil

tnyir metal

health food additive

vincristine

antineoplastic

vinblastine

antineoplastic

vindesine

antineoplastic

vitamin A

mega doses

vitamin D

mega doses

In general, neurotoxicity is dose-related, and presents as reduced intestinal
motility and peripheral neuropathy, especially in the distal muscles of the hands and feet,
postural hypotension, and atony of the urinary bladder. Similar problems have been

5 reported with taxol and cisplatin (Mollman, J. E., 1990, New Eng Jour Med.

322:126-127), although cisplatin-related neurotoxicity reportedly can be alleviated with
nerve growth factor (NGF) (Apfel, S. C, et al, 1992, Annals of Neurology 3 1 :76-80).
Although the neurotoxicity is sometimes reversible after removal of the neurotoxic agent,
recovery reportedly can be a very slow process (Legha, S., 1986, Medical Toxicology

10 1:421-427; Olesen, et al., 1991, Drug Safety 6:302-314).

Exemplary neuropathies associated with a systemic condition include: uremia,
childhood cholestatic liver disease, chronic respiratory insufficiency, alcoholic
polyneuropathy, multiple organ failure, sepsis, hypoalbuminemia, eosinophilia-myalgia
syndrome, hepatitis, porphyria, hypoglycemia, vitamin deficiency, chronic liver disease,

15 primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme disease, herpes zoster,

Guillain-Barre syndrome, chronic inflanunatory demyelinating polyradiculoneuropathy,
sensory perineuritis, acquired immunodeficiency syndrome (AIDS)-associated
neuropathy, Sjogren's syndrome, primary vasculitis (such as polyarteritis nodosa),
allergic granulomatous angiitis (Churg-Strauss), hypersensitivity angiitis, Wegener's

20 granulomatosis, rheumatoid arthritis, systemic lupus erythematosis, mixed connective
tissue disease, scleroderma, sarcoidosis, vasculitis, systemic vasculitides, acute
inflammatory demyelinating polyneuropathy, post-polio syndrome, carpal tunnel

wo 00/62798

.19-

PCT/US00/0976S

syndrome, pandysautonomia, primaiy systemic amyloidosis, hypothyroidism, chronic
obstructive pulmonary disease, acromegaly, malabsorption (sprue, celiac disease),
carcinomas (sensory, sensorimotor, late and demyelinating), lymphoma (including
Hodgkin's), polycythemia vera, multiple myeloma (lytic type, osteosclerotic, or solitary
5 plasmacytoma), benign monoclonal gammopathy, macroglobulinemia, and
cryoglobulinemia.

Exemplary genetically acquired neuropathies include: peroneal muscular atrophy
(Charcot-Marie-Tooth Disease, types I, II, and X), hereditary amyloid neuropathies,
hereditary sensory neuropathy (type I and type II), porphyric neuropathy, hereditary

10 liability to pressure palsy, Fabry's disease, adrenomyeloneuropathy, Riley-Day
syndrome, Dejerine-Sottas neuropathy (hereditary motor-sensory neuropathy-III),
Refsum's disease, ataxia-telangiectasia, hereditary tyrosinemia, anaphalipoproteinemia,
abetalipoproteinemia, giant axonal neuropathy, metachromatic leukodystrophy, globoid
cell leukodystrophy, and Friedrich's ataxia. Also included in the invention are

15 mononeuropathy multiplex, plexopathy, and pure motor neuropathy.

The angiogenic growth factors of the invention are administered in effective
amoxmts. An effective amount is a dosage of the angiogenic growth factor nucleic acid
sufficient to provide a medically desirable result. The effective amount will vary with the
particular condition being treated, the age and physical condition of the subject being

20 treated, the severity of the condition, the duration of the treatment, the nature of the

concurrent therapy (if any), the specific route of administration and like factors within the
knowledge and expertise of the healthcare practitioner. For example, in connection with
peripheral neuropathy, an effective amount is that amoimt which alleviates a symptom of
the neuropathy. Likewise, an effective amount for treating a subject who has sustained a

25 peripheral nerve injury, would be an amount sufficient to enhance peripheral nerve
regeneration. Thus, it will be understood that the angiogenic growth factor of the
invention can be used to treat the above-noted conditions prophylactically in subjects at
risk of developing the foregoing conditions. As used herein, "treat" embraces all of the
foregoing. It is preferred generally that a maximum dose be used, that is, the highest safe

30 dose according to sound medical judgment.

A particularly important aspect of the invention involves the use of the angiogenic
growth factors of the invention for treating subjects who have sustained a peripheral

wo 00/62798 PCT/US00/d9765

-20-

neuropathy as a side effect of ischemic heart disease. Ischemia refers to a lack of oxygen
due to inadequate perfusion of blood. Ischemic heart disease is characterized by a
disturbance in cardiac function due to an inadequate supply of oxygen to the heart. The
most common form of this disease involves a reduction in the lumen of coronary arteries,
5 which limits coronary blood-flow.

When ischemic heart disease becomes very serious, management of the disease
becomes invasive. Until recently, ischemic heart disease was treated by coronary-artery,
bypass surgery. Less invasive procedures, however, now have been developed. These
procedures involve the use of catheters introduced into the narrowed region of the blood

10 vessel ("the stenosis") for mechanically disrupting, laser ablating or dilating the stenosis.
The most widely used method to achieve revascularization of a coronary artery is
percutaneous transluminal coronary angioplasty. A flexible guide wire is advanced into a
coronary artery and positioned across the stenosis. A balloon catheter then is advanced
over the guide wire until the balloon is positioned across the stenosis. The balloon then is

15 repeatedly inflated until the stenosis is substantially eliminated. This procedure, as

compared to heart surgery, is relatively noninvasive and typically involves a hospital stay
of only a few days. The procedure is an important tool in the management of serious
heart conditions and can also be used to deliver the angiogenic growth factor of the
invention to a local site of ischemic tissue and for treatment of a neuropathy in the

20 ischemic tissue. Alternatively, the angiogenic growth factors can be intramuscularly

injected directly into the ischemic tissue. In certain embodiments, the angiogenic growth
factors are in the form of controlled release preparations for the sustained delivery of the
factors to the ischemic or other tissue that presents symptoms of a peripheral neuropathy.
Controlled release systems for delivery of an angiogenic growth factor nucleic acid or

25 polypeptide are described in more detail below.

A subject, as used herein, refers to any mammal (preferably, a human) that may
be susceptible to a condition associated with peripheral neuropathy (such as the
conditions described above). In certain embodiments, the manunal is otherwise free of
symptoms calling for angiogenic growth factor treatment. Different aspects of the

30 invention may exclude one or more of the following subject populations that present with
a peripheral neuropathy: (1) patients presenting with a vascular disease; (2) patients
presenting with a vascular obstruction (large vessels); (3) patients presenting with a

wo 00/62798 PCT/US00/0976S

-21-

microvascular disease; (4) patients presenting with an ischemic tissue, such as an
ischemic limb; (5) patients being treated with an angiogenic growth factor to promote
revascularization; and (6) patients who are being treated using gene therapy.

In some particular embodiments, the preferred vehicle is a biocompatible micro
5 particle or implant that is suitable for implantation into the subject. Exemplary

bioerodible implants that are useful in accordance with this method are described in PCT
International application no. PCTAJS/03307 (Publication No. WO 95/24929, entitled
"Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no.
213,668, filed March 15, 1994). PCT/US/0307 describes a biocompatible, preferably

10 biodegradable polymeric matrix for containing an exogenous gene under the control of an
appropriate promotor. The polymeric matrix is used to achieve sustained release of the
exogenous gene in the subject. In accordance with the instant invention, the angiogenic
growth factor nucleic acids described herein are encapsulated or dispersed within the
biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.

15 The polymeric matrix preferably is in the form of a micro particle such as a micro sphere
(wherein the angiogenic growth factor nucleic acid is dispersed throughout a solid
polymeric matrix) or a microcapsule (wherein the angiogenic growth factor nucleic acid
is stored in the core of a polymeric shell). Other forms of the polymeric matrix for
containing the angiogenic growth factor nucleic acid include films, coatings, gels,

20 implants, and stents. The size and composition of the polymeric matrix device is selected
to result in favorable release kinetics in the tissue into which the matrix device is
implanted. The size of the polymeric matrix devise further is selected according to the
method of delivery which is to be used, typically injection into a tissue or administration
of a suspension by aerosol into the nasal and/or pulmonary areas. The polymeric matrix

25 composition can be selected to have both favorable degradation rates and also to be

formed of a material which is bioadhesive, to further increase the effectiveness of transfer
when the devise is administered to a vascular surface. The matrix composition also can
be selected not to degrade, but rather, to release by diffusion over an extended period of
time.

30 Both non-biodegradable and biodegradable polymeric matrices can be used to

deliver the angiogenic growth factor nucleic acids of the invention to the subject.
Biodegradable matrices are preferred. Such polymers may be natural or synthetic

wo 00/62798

-22-

PCT/USOO/09765

polymers. Synthetic polymers are preferred. The polymer is selected based on the period
of time over which release is desired, generally in the order of a few hours to a year or
longer. Typically, release over a period rangmg from between a few hours and three to
twelve months is most desirable. The polymer optionally is in the form of a hydrogel that
can absorb up to about 90% of its weight in water and further, optionally is cross-linked
with multi-valent ions or other polymers.

In general, the angiogenic growth factor nucleic acids of the invention are
delivered using the bioerodible implant by way of diffusion, or more preferably, by
degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to
form the biodegradable delivery system include: polyamides, polycarbonates,
polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,
polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,
polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro
celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl
cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose
acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium
salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexyhnethacrylate), poly(isodecyl methacrylate),
poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate),
poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and
polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,
poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such as
polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,
polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and
natural polymers such as alginate and other polysaccharides including dextran and
cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical

wo 00/62798

-23-

PCT/USOO/09765

groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein
and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In
general, these materials degrade either by enzymatic hydrolysis or exposure to water in

5 vivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodible hydrogels
described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, 1993, 26,
581-587, the teachings of which are incorporated herein, poly hyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl

10 methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acryiate), poly(isopropyl
acrylate), poly(isobutyl acryiate), and poly(octadecyl acryiate). Thus, the invention
provides a composition of the above-described angiogenic growth factors for use as a

15 medicament, methods for preparing the medicament and methods for the sustained

release of the medicament in vivo. In the preferred embodiments, the angiogenic growth
factor nucleic acid is a human VEGF nucleic acid, alone or in combination v^th a human
bFGF nucleic acid. Preferably, the angiogenic growth factor nucleic acid is operably
linked to a gene expression sequence to permit expression of the angiogenic growth

20 factor nucleic acid in the target cell.

Compaction agents also can be used alone, or in combination with, a vector of the
invention. A "compaction agent", as used herein, refers to an agent, such as a histone,
that neutralizes the negative charges on the nucleic acid and thereby permits compaction
of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the

25 uptake of the nucleic acid by the target cell. The compaction agents can be used alone,
i.e., to deliver the isolated angiogenic growth factor nucleic acid in a form that is more
efficiently taken up by the cell or, more preferably, in combination with one or more of
the above-described vectors.

Other exemplary compositions that can be used to facilitate uptake by a target cell

30 of the angiogenic growth factor nucleic acids include calcium phosphate and other
chemical mediators of intracellular transport, microinjection compositions,
electroporation and homologous recombination compositions (e.g., for integrating an

wo 00/62798

-24-

PCT/USOO/09765

angiogenic growth factor nucleic acid into a preselected location within the target cell
chromosome).

The angiogenic growth factor nucleic acids code for an angiogenic growth factor
polypeptide. As used herein, a "angiogenic growth factor polypeptide" refers to a
5 polypeptide that, either directly or indirectly, enhances endothelial cell growth.

Angiogenic growth factor polypeptides are useful for alleviating a symptom of peripheral
neuropathy and/or enhancing nerve regeneration. The preferred angiogenic growth factor
polypeptides of the invention are the human VEGF isoforms, administered alone or in
combination with human bFGF. Angiogenic growth factor polypeptides further embrace

10 functionally equivalent variants, and analogs of angiogenic growth factors, provided that
the fragments, variants, and analogs alleviate a symptom of a peripheral neuropathy
and/or enhance nerve regeneration. The invention also embraces proteins and peptides
coded for by any of the foregoing angiogenic growth factor nucleic acids. The invention
also embraces agents that upregulate expression of an angiogenic growth factor

1 5 polypeptide in vivo .

A "functionally equivalent variant" of an angiogenic growth factor is capable of
alleviating a symptom of a peripheral neuropathy and/or enhancing nerve regeneration in
vitro or in vivo. An in vitro assay or an in vivo animal model (see, e.g., the Examples)
can be used as a screening assay to measure the ability of a polypeptide to alleviate a

20 symptom of a peripheral neuropathy and/or enhance nerve regeneration. The animal
model disclosed in the Examples can be used to screen therapeutic drugs because it is
predictive of the ability of the polypeptide to treat a peripheral neuropathy in vivo.
Exemplary "functionally equivalent variants" of the angiogenic growth factors (such as
the exemplary growth factors disclosed herein) include fragments of these factors, as well

25 as polypeptide analogs of these factors which contain conservative amino acid

substitutions, provided that the polypeptide variants and analogs are capable of alleviating
a symptom of a peripheral neuropathy and/or enhancing nerve regeneration.

It will be appreciated by those skilled in the art that various modifications of the
angiogenic growth factor polypeptide having the sequences deposited in the publicly

30 available databases or functionally equivalent fi-agments thereof can be made v^dthout
departing from the essential nature of the invention. Accordingly, it is intended that
polypeptides which have the published amino acid sequences but which include

wo 00/62798 PCT/USOO/09765

-25-

conservative substitutions are embraced within the instant invention. As used herein,
"conservative amino acid substitution" refers to an amino acid substitution which does
not alter the relative charge or size characteristics of the polypeptide in which the amino
acid substitution is made. Conservative substitutions of amino acids include substitutions
5 made amongst amino acids with the foUov^ng groups: (1) M,I,L,V; (2) F,Y,W; (3)
K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D. Fusion proteins, in which a peptide of
the invention is coupled to a solid support (such as a polymeric bead for controlled
release), or a reporter group (such as radiolabel or other tag), also are embraced within
the invention.

10 When used therapeutically, the isolated angiogenic growth factors of the invention

are administered in therapeutically effective amounts. In general, a therapeutically
effective amount means that amount necessary to delay the onset of, inhibit the
progression of, or halt altogether the particular condition being treated. As noted above, a
therapeutically effective amount will vary with the subject's age, condition, and sex, as

15 well as the nature and extent of the disease in the subject, all of which can be determined
by one of ordinary skill in the art. The dosage may be adjusted by the individual
physician or veterinarian, particularly in the event of any complication. A therapeutically
effective amoimt can vary throughout a broad dosage range, e.g., from 0,01 mg/kg to
about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most

20 preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations
daily, for one or more days. In certain aspects of the invention described above, the
angiogenic growth factors are administered over a period of months to years in the case
of a chronic peripheral neuropathy.

The therapeutically effective amount of the isolated angiogenic growth factor is

25 that amount effective to inhibit the development of peripheral neuropathy, alleviate a
symptom of a peripheral neuropathy, and/or enhance nerve regeneration as determined
by, for example, standard tests known in the art. It is believed that the angiogenic grov^
factors directly and/or indirectly enhance nerve regeneration in the vicinity of the target
cell. Diagnostic tests that are used to diagnose a peripheral neuropathy can by used to

30 select an effective amount of the angiogenic growth factor. In vitro assays are available
to determine whether a factor has been effective in inducing nerve regeneration.

wo 00/62798

.26-

PCTAJS00/0976S

Optionally, the isolated angiogenic growth factor is administered to the subject in
combination with an alternative method for treating the neuropathy or for treating the
particular condition that is associated with the peripheral neuropathy. See, e.g.,
Harrisons, Principles of Internal Medicine (McGraw Hill, Inc., New York) for a
5 description of standard treatments for peripheral neuropathies and/or for conditions that
also present with symptoms of a peripheral neuropathy. The method for treating
neuropathy may be a surgical method, a drug for treating neuropathy, a gene therapy
method or a combination of the foregoing.

Surgical methods for treating a condition of vascular insufficiency include

10 procedures such as bypass surgery, atherectomy, laser procedures, ultrasonic procedures,
and balloon angioplasty to enhance vascularization of an ischemic tissue. In certain
embodiments, the isolated angiogenic growth factor is administered to a subject in
combination with a balloon angioplasty procedure. The isolated angiogenic grov^ factor
is attached to the balloon angioplasty catheter in a manner which permits release of the

15 isolated angiogenic growth factor at the site of the atherosclerotic plaque. The isolated
angiogenic growth factor may be attached to the balloon angioplasty catheter in
accordance with standard procedures known in the art. See, e.g., U.S. Patent No.
5,652,225, entitled "Methods and products for nucleic acid delivery", issued to J. Isner,
for a description of a balloon angioplasty procedure for delivering VEGF.

20 Additionally, the angiogenic growth factor may be administered in combination

with the toxic agent which causes the neuropathy, e.g., a neoplastic agent, to alleviate the
symptoms of peripheral neuropathy that are a side effect of the neoplastic agent.

The angiogenic growth factor also may be administered in combination with a
drug for treating the condition which is believed to be associated with, or caused by, the

25 peripheral neuropathy. For example, the angiogenic growth factor may be administered
in combination with a drug for treating a diabetic condition (e.g., insulin), to alleviate the
symptoms of peripheral neuropathy that are a side effect of the diabetic condition.

The above-described drug therapies are well known to those of ordinary skill in
the art and are administered by modes known to those of skill in the art. The drug

30 therapies are administered in amounts which are effective to achieve the physiological
goals (to prevent or reduce the physiological consequences of a peripheral neuropathy), in
combination with the isolated angiogenic growth factor(s) of the invention. Thus, it is

wo 00/62798 PCT/US00/0y765

-27-

contemplated that the drug therapies may be administered in amounts which are not
capable of preventing or reducing the physiological consequences of the peripheral
neuropathy when the drug therapies are administered alone but which are capable of
preventing or reducing the physiological consequences of the peripheral neuropathy when
5 administered in combination with the isolated angiogenic growth factors of the invention.
The isolated angiogenic growth factor may be administered alone or in
combination with the above-described drug therapies as part of a pharmaceutical
composition. Such a pharmaceutical composition may include the isolated angiogenic
growth factor in combination with any standard physiologically and/or pharmaceutically

10 acceptable carriers which are known in the art. The compositions should be sterile and
contain a therapeutically effective amount of the isolated angiogenic growth factor in a
unit of weight or volume suitable for administration to a patient. The term
"pharmaceutically-acceptable carrier" as used herein means one or more compatible solid
or liquid filler, diluents or encapsulating substances which are suitable for administration

15 into a human or other animal. The tenn "carrier" denotes an organic or inorganic

ingredient, natural or synthetic, with which the active ingredient is combined to facilitate
the application. The components of the pharmaceutical compositions also are capable of
being co-mingled with the molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially impair the desired

20 pharmaceutical efficacy. Pharmaceutically acceptable fiirther means a non-toxic material
that is compatible vrfth a biological system such as a cell, cell culture, tissue, or organism.
The characteristics of the carrier will depend on the route of administration.
Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts,
buffers, stabilizers, solubilizers, and other materials which are well known in the art.

25 Compositions suitable for parenteral administration conveniently comprise a

sterile aqueous preparation of the angiogenic growth factors, which is preferably isotonic
with the blood of the recipient. This aqueous preparation may be formulated according to
known methods using suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation also may be a sterile injectable solution or suspension in a

30 non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-
butane did. Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed

wo 00/62798 PCT/USOO/09765

-28-

oils are conventionally employed as a solvent or suspending medium. For this purpose,
any bland fixed oil may be employed including synthetic mono- or di-glycerides. In
addition, fatty acids such as oleic acid may be used in the preparation of injectables.
Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc.
5 administrations can be foimd in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, PA.

A variety of administration routes are available. The particular mode selected will
depend, of course, upon the particular drug selected, the severity of the condition being
treated, and the dosage required for therapeutic efficacy. The methods of the invention,

10 generally speaking, may be practiced using any mode of administration that is medically
acceptable, meaning any mode that produces effective levels of the active compoimds
without causing clinically unacceptable adverse effects. Intramuscular administration is
preferred; however other modes of administration may be acceptable including, e.g.,
other parenteral routes, oral, rectal, topical, nasal, or interdermal. The term "parenteral"

15 includes subcutaneous, intravenous, intramuscular, or infusion.

The pharmaceutical compositions may conveniently be presented in unit dosage
form and may be prepared by any of the methods well-known in the art of pharmacy. All
methods include the step of bringing the angiogenic growth factors into association with a
carrier which constitutes one or more accessory ingredients. In general, the compositions

20 are prepared by uniformly and intimately bringing the angiogenic growth factors into
association with a liquid carrier, a finely divided solid carrier, or both, and then, if
necessary, shaping the product.

Other delivery systems can include time-release, delayed release or sustained
release delivery systems such as those described in detail above. In general, such systems

25 can avoid repeated administrations of the angiogenic growth factors, increasing

convenience to the subject and the physician. Many types of release delivery systems are
available and known to those of ordinary skill in the art. They include the above-
described polymeric systems, as well as polymer base systems such as poly(lactide-
glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,

30 polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers
containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems
also include non-polymer systems that are: lipids including sterols such as cholesterol.

wo 00/62798 PCTAJSOO/09765

-29-

cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; sylastic systems; peptide based systems; wax coatings;
compressed tablets using conventional binders and excipients; partially fused implants;
and the like. Specific examples include, but are not limited to: (a) erosional systems in

5 which the angiogenic growth factor is contained in a form within a matrix such as those
described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b)
diffiisional systems in which an active component permeates at a controlled rate from a
polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In addition,
pump-based hardware delivery systems can be used, some of which are adapted for

10 implantation.

Use of a long-term sustained release implant may be particularly suitable for
treatment of chronic conditions. Long-term release, are used herein, means that the
implant is constructed and arranged to delivery therapeutic levels of the active ingredient
for at least 30 days, and preferably 60 days. Long-term sustained release implants are
15 well-known to those of ordinary skill in the art and include some of the release systems
described above.

The isolated angiogenic growth factor may be administered alone or in
combination with the above-described drug therapies by any conventional route,
including injection or by gradual infusion over time. The administration may, for

20 example, be intramuscular, intravenous, intraperitoneal, intra-cavity, subcutaneous, oral,
or transdermal. When using the isolated angiogenic growth factor of the invention, direct
administration to the nerve injury site, such as by administration in conjunction with a
balloon angioplasty catheter or intramuscular injection, is preferred.

Preparations for parenteral administration include sterile aqueous or non-aqueous

25 solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media. Parenteral vehicles
include sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride,

30 lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient

replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the

wo 00/62798 PCT/USOO/09765

-30-

like. Preservatives and other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

In general, the angiogenic grovslh factor nucleic acids can be administered to the
subject (any mammalian recipient) using the same modes of administration that currently
5 are used for gene therapy in humans (e.g., adenovirus-mediated gene therapy).

Preferably, the angiogenic growth factor nucleic acid (contained in, or associated wdth, an
appropriate vector) is administered to the mammalian recipient by balloon angioplasty
catheter (described above), or intra-vascular, or, more preferably, intramuscular injection.
A procedure for performing in vivo gene therapy for delivering a nucleic acid encoding
10 an intact angiogenic grov^ factor (VEGF) to cells in vivo for treating a vascular injury is
reported in U.S. Patent No. 5,830,879, entitled "Treatment of vascular injury using
vascular endothelial growth factor", issued to J. Isner.

As an illustrative example, a vector containing a VEGF nucleic acid is delivered
to a site of vascular injury in a subject who is a candidate for such gene therapy. Then,
15 the vector genetically modifies the target cells (e.g., endothelial cells, muscle cells, nerve
cells) in vivo v^th DNA (RNA) encoding the VEGF. Such genetically modified cells
express VEGF which is believed to directly or indirectly alleviate the symptoms of a
peripheral neuropathy in vivo. Although not wishing to be bound to a particular theory
or mechanism, it is believed that such genetically modified cells exhibit enhanced nerve
20 regeneration in vitro and in vivo.

The invention will be more fully understood by reference to the following
examples. These examples, however, are merely intended to illustrate the embodiments
of the invention and are not to be construed to limit the scope of the invention.

EXAMPLES

25 Introduction:

The Examples employ certain procedures which are known in the art and which
may have been published in issued U.S. patents or foreign patent documents, including
the following documents:

Patent/Serial/Publication
No.

Title

Inventor

U.S. 5,830,879

Treatment of vascular injury
using vascular endothelial
growth factor

J. Isner

wo 00/62798 PCTAJSOO/09765

-31-

U.S. 5,652,225

Methods and products for
nucleic acid delivery

J. Isner

U.S. Serial No. 08/545,998

Method for Treating
Ischemic Tissue

J. Isner

U.S. 5,648,335;
U.S. 5,633,228; and
U.S. 5,569,648

Prevention and treatment of
peripheral neiu-opathy

M. Lewis, et al.

Selected methods that previously have been described in the above-identified
patent documents and that can be used in accordance with the methods of the instant
invention are briefly summarized herein.

5 Method; Plasmids

Complementary DNA clones for recombinant human VEFG]65 isolated from
cDNA libraries prepared from HL60 leukemia cells, were assembled into a simple
eukaryotic expression plasmid that utilizes 736 bp cytomegalovirus promoter/enhancer to
drive VEGF expression. Downstream from the VEGF cDNA is an SV40 polyadenylation

10 sequence. Also included in this plasmid is a fragment containing the SV40 origin of
replication that includes the 72 bp repeat, but this sequence is not fiinctionally relevant
(for autonomous replication) in the absence of SV40 T antigen. These fragments occur in
the pUCl 1 8 vector which includes an E. Coli origin of replication and the P-galactosidase
gene for ampicillin resistance. The biological activity of VEGF165 secreted from cells

15 transfected wdth this construct (phVEGFies) was previously confirmed by evidence that
media conditioned by transfected human 293 cells promoted the proliferation of capillary
cells (Leung, et al. Science, 246:1306-9 (1989)).

The plasmid pGSVLacZ (courtesy of Dr. Claire Bonnerot) containing a nuclear
targeted P-galactosidase sequence coupled to the simian virus 40 early promoters

20 (Bonnerot, et al., Proa Natl AcadSci, US.A., 84:6795-9 (1987)) was used for all the
control transfections.
Method: Animal Model

New Zealand white rabbits with operatively induced unilateral hindlimb vascular
insufficiency, (Takeshita, et al., Circulation, 90:228-234 (1994); Takeshita, et al., J, Clin,

IS Invest, 93:662-70 (1994); Pu, et al.. Circulation, 88:208-215 (1993), were used to model
both acute and chronic ischemia. All protocols were approved by the Institutional
Animal Care and Use Committee. The care of animals complied with the guidelines of

wo 00/62798

"32-

PCTAJSOO/09765

the Canadian Council of Animal Care, the Principles of Laboratory Animal Care, and the
Guide for the Care and Use of Laboratory Animals (NIH publication No. 80-23, revised
1985). Fifty-nine male New Zealand White rabbits (mean weight = 3 kg) were
anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). Through a longitudinal

5 incision performed in a medial thigh, the femoral artery was dissected free along its entire
length, as were all major branches of the femoral artery, including the inferior epigastric,
deep femoral, lateral circumflex and superficial epigastric arteries. After ftirther
dissecting the popliteal and saphenous arteries distally, the external iliac artery as well as
all of the above arteries were ligated. Finally, the femoral artery was completely excised

10 from its proximal origin as a branch of the external iliac artery to the point distally where
it bifiarcates into the saphenous and popliteal arteries.
Method; Intramuscular (IM) Gene Transfer

Acute Limb Ischemia, Twenty-eight rabbits were used to study the impact of IM
gene transfer on acute hindlimb ischemia. Immediately following femoral artery excision

15 as outlined above, five different sites in three major thigh muscles were injected directly
with plasmid DNA using a 3 ml syringe and 27-gauge needle advanced through a small
skin incision. For each injection, the tip of the needle was inserted into the adductor (2
sites), medial large (2 sites), and semimembranous muscles; care was taken, by directly
visualizing each muscle during the injection, to avoid penetrating the muscle with the

20 injectate. To the same end, the rate of injection was in each case slowed to approximately
5 seconds so that injected solution would not leak through the epimysium. This injection
technique was used to administer a total volume of 2.5 ml of a) a 500 ng ph VEGF165 in
saline (n+8; b) 500 ^ig phVEGFi65 in 0.75% bupivacaine, previously shovm to enhance
transgene uptake by striated muscle (n+10) (Danko I, Gene Therapy, 1:1 14-21 (1994); or

25 c) 500 jxg pGSVLacZ encoding nuclear targeted p-galactosidase (n+10). After

completing 5 injections (0.5 ml @ for each animal), the skin was then closed using 4.0
nylon.

Chronic Limb Ischemia. Thirty-one rabbits were used to study the effects of IM
gene therapy for chronic hindlimb ischemia. The sole distinction between the chronic
30 ischemia model and model of acute limb ischemia described above, is that an interval of
10 days was permitted for post-operative recovery, including development of endogenous
collateral vessels. Accordingly, 10 days following femoral artery excision, the rabbits

wo 00/62798

PCT/USOO/09765

"33-

were returned to the catherization laboratory. After completing baseline physiological
measurements described below, IM gene transfer using the identical technique described
above was performed with a) 500 \ig phVEGFies diluted in 2.5 ml of saline (n=8); b) 500
Jig phVEGFi65 diluted in 0.75% bupivacame (n=8); c) 500 ng of pGSVLacZ diluted in
5 2.5 ml of saline; or d) 500 iig of pGSVLacZ diluted in 2.5 ml of 0.75% bupivacaine
(n=8). In each case, after completing all 5 injections, the skin was closed as above.

VEGF Gene Expression in Skeletal Muscle. To evaluate expression of
phVEGFi65 gene in skeletal muscle, sixteen additional male New Zealand white rabbits
from both acute and chronic ischemia models (2 rabbits at each time point) were

10 sacrificed at 3, 7, 14, and 30 days post-transfection. The presence of human VEGF

mRNA was detected using reverse transcription-polymerase chain reaction (RT-PCR) as
previously described (Takeshita, et al., Proc Natl Acad Sci). To ensure specificity and
avoid amplification of endogenous rabbit VEGF, primers were selected fi-om a region
which is not conserved among different species. Sequences of primers used were: 5'-

15 GAGGGCAGAATCATCACGAAGT-3' (sense) (SEQ ID NO: 1): 5'-

TCCTATGTGCTGGCCTTGGTGA-3' (antisense) (SEQ ID NO:2). RT-PCR products
were analyzed by 2% agarose gel electrophoresis. DNA bands were visualized imder UV
illumination after staining with ethiditun bromide.

EXAMPLE l i Define certain morphologic, temporal, and functional aspects of
20 therapeutic angiogenesis.

A. Cellular proliferation contributing to the development of the nascent
collateral circulation is augmented in ischemic limbs in response to therapeutic
angiogenesis.

We determined the extent to which proliferative activity of vascular cells is
25 augmented during therapeutic angiogenesis with VEGF (Takeshita S, et al., Am J Pathol
1995;147:1649-1660). Ten days following induction of limb ischemia by surgically
excising femoral artery of rabbits, either VEGF (500-1,000 mg) or saline was
administered as a bolus into the iliac artery of the ischemic limb. Cellular proliferation
was evaluated by bromodeoxyuridine (BrDU) labeling for 24 hrs at day 0 (immediately
30 prior to VEGF administration) and at days 3, 5, and 7 post- VEGF. EC proliferation in the
midzone collaterals of VEGF-treated animals increased 2.8-fold at day 5 (p<0.05, vs.
control), and returned to baseline levels by day 7. Smooth muscle cell proliferation in

wo 00/62798

.34.

PCTAJSOO/09765

midzone collaterals also increased 2.7-fold in response to VEGF (p<0.05). No significant
increase in cellular proliferation was observed in either the stem or re-entry collaterals.
Reduction of hemodynamic deficit in the ischemic limb measxired by lower limb blood
pressure was docxmiented at day 7 post- VEGF (p<0.01, vs. control). These data thus
5 support the concept that increased cellular proliferation contributes to the formation of
collateral vessels following therapeutic angiogenesis with VEGF.

Subsequently, we have similarly documented the time-course of cellular
proliferative activity in a mouse model of hindlimb ischemia (CoxxfBnhal T., et al., Am J
Pathol 1998;152:1667-1679). The femoral artery of one hindlimb was ligated and

1 0 excised. Laser Doppler perfusion imaging (LDPI) was employed to document the
consequent reduction in hindlimb blood flow which typically persisted for up to seven
days. Serial in vivo examinations by LDPI disclosed that hindlimb blood flow was
progressively augmented over the course of 14 days, ultimately reaching a plateau
between 21 and 28 days, Morphometric analysis of capillary density performed at the

15 same timepoints selected for in vivo analysis of blood flow by LDPI confirmed that the
histologic sequence of neovascularization corresponded temporally to blood flow
recovery detected in vivo. EC proliferation was documented by immimostaining for
BrdU injected 24 hrs prior to each of these timepoints, providing further evidence that
angiogenesis constitutes the basis for improved collateral-dependent flow in this animal

20 model. BrdU staining was performed in the ischemic versus normal hindlimbs.

Proliferative activity peaked at 7 days (1235 ±254 vs 8±14 BrdU-positive cells/mm^ for
the ischemic vs normal limbs respectively (p<0.001); proliferative activity was then
subsequently reduced at days 1 4 and 2 1 . Double immunolabeling for BrdU and CD-3 1
demonstrated proliferating ECs in the ischemic limb. Most proliferating ECs localized to

25 small capillaries, although EC proliferation was observed in small arteries as well.

Capillary density and proliferative activity were also examined in mice treated with PF-4
and sacrificed 14 days after surgery. A significant decrease in capillary density (268±1 95
vs 1053±371 capilla^ies/mm^ p<0.01) and EC proliferation (16±29 vs 935±239 BrdU-
positive cells/mm^, p<0.01) were found in PF-4 vs PBS-injected mice respectively.

30 Finally, in two patients who underwent amputations followdng VEGF gene

therapy, EC proliferative activity in the ischemic limb has been documented as well.

wo 00/62798 PCTAJSOO/09765

-35-

using double immunostaining for PCNA and CD31 (Baumgartner I, et al., Circulation
1998;97:1 1 14-1 123) (Isner JM. et al.. Lancet 1996;348:370-374).

B. Therapeutic angiogenesis may be employed successfully to supplement
established, native collateral circulation.
5 Long-term experiments regarding this issue are still on-going. In the interim,

however, we have documented by angiographic analysis supplemental collateral vascular
development in patients with collateral vessels that had developed endogenously prior to
gene transfer (Baumgartner I, et al., Circulation 1998;97:1 1 14-1 123) (Isner JM, et al., J
Vase Surg 1998;28:964-975). Thus the extent of collateral development appears to be
10 influenced less by the extent of pre-existing collaterals than by other possible
determinants.

C* Therapeutic angiogenesis preserves receptor-mediated endothelium-
dependent flow in the rabbit ischemic hindlimb.

Disturbed endothelium-dependent blood flow has been previously shown to be a

15 feature of native collateral vessels. We therefore investigated the hypothesis that

administration VEGF may promote recovery of disturbed endothelium-dependent blood
flow in our rabbit model of hindlimb ischemia (Bauters C, et al., Circulation
1995;91:2802-2809).

Ischemia was induced by ligation of external iliac artery and excision of femoral

20 artery in one limb of NZW rabbits (day 0). Flow velocity was measured using a Doppler
guidewire at rest and following serotonin and acetylcholine. Blood flow (ml/min) was
calculated assuming a circular lumen geometry. In untreated control animals with an
ischemic limb, serotonin administered at days 10 or 40 produced a decrease in hindlimb
blood flow (71 ±2% and 33±6% reduction from baseline, respectively); in contrast, among

25 animals treated with a single bolus dose of VEGF administered selectively into the
internal iliac artery at day 10 and studied at day 40, serotonin produced an increase in
flow (1 19±8% fi-om baseline; p<0.05 vs controls). Acetylcholine induced only a moderate
increase in flow in control animals (152±15% at day 10, 177±14% at day 40), in contrast
to a profound increase among VEGF-treated animals studied at day 40 (254±25%; p<0.05

30 vs controls).

To our knowledge, these findings constitute the first demonstration of successful
pharmacologic modulation of disturbed endothelium-dependent flow in the arterial

wo 00/62798 PCTA;S00/09765

-36-

circulation subserved by collateral vessels. This physiologic benefit complements
previously reported anatomic findings suggesting a favorable impact of angiogenic
growth factors on collateral-dependent limb ischemia.

EXAMPLE 2 : Investigation of certain conditional factors which may modulate the
5 outcome of therapeutic angiogenesis.

A. Hypoxia modulates the response to angiogenesis induced by VEGF and

bFGF.

To evaluate this hypothesis, we investigated whether low oxygen tension or
cytokines known to promote neovascularization in vivo could modulate the expression of

10 VEGF or bFGF in human vascular smooth muscle cells (SMCs) (Brogi E, et al..

Circulation 1994;90:649-652). SMCs were treated with platelet derived growth factor BB
(PDGF BB) or transforming grov^ factor pi (TGF-pi) or exposed to low oxygen
tension in serum-free medium. Northern analysis detected low basal levels of VEGF and
bFGF mRNA in extracts of unstimulated SMCs. However, both VEGF and bFGF

15 transcripts increased follov^dng administration of PDGF BB (10 or 20 ng/ml) or TGF-pi
(0. 1 or 1 ng/ml). Hypoxia was a potent stimulus for VEGF gene expression, but had no
apparent effect on bFGF steady state mRNA levels. These results documented that certain
indirect angiogenic cytokines, such as PDGF BB or TGF-pi, may act via induction of
bFGF and VEGF gene expression in cells resident near ECs in vivo. Hypoxia constitutes

20 a potent stimulus for VEGF gene expression, but does not regulate bFGF under the same
experimental conditions.

We further investigated whether the role of human ECs might, under selected
conditions, extend beyond that of a target to involve contingency synthesis of VEGF
(Namiki A, et al., J Biol Chem 1995;270:31189-31 195). In both unstimulated human

25 umbilical vein ECs (HUVECs) or human derma-derived microvascular ECs (HMECs),
Northern analysis detected no VEGF transcripts. Phorbol-12-myristate 13-acetate (PMA,
10*' M) treatment, however, induced VEGF mRNA expression in both HUVECs and
HMECs, peaking at 3hrs and 6 hrs respectively, and returning to undetectable levels by
12 hrs. In vitro exposure of HUVECs to an hypoxic environment (p02=35 nrniHg) for

30 12, 24 and 48 hrs, and HMECs for 6, 12, 24 and 48 hrs induced VEGF mRNA in a time-
dependent fashion. Re-exposure to normoxia (p02=l 50 mmHg) for 24 hrs after 24 hrs of
hypoxia returned VEGF mRNA transcripts to imdetectable levels in HUVECs. Cobalt

wo 09/62798 PCT/USOO/09765

-37-

chloride (Co^"*^and nickel chloride (Ni^**") treatment each induced VEGF mRNA in ECs.
Cycloheximide treatment further augmented expression of VEGF mRNA induced by
Co^"*", Ni^"^ and hypoxia in HUVECs. VEGF protein production in hypoxic HUVECs was
demonstrated immunohistochemically. Conditioned media from hypoxic HUVECs
5 caused a two-fold increase in the incorporation of tritiated thymidine. Finally, immune
precipitates of anti-KDR probed with anti-P-tyr antibodies demonstrated evidence of
receptor autophosphorylation in hypoxic, but not normoxic, HUVECs. These findings
thus established the potential for an autocrine pathway that may augment and/or amplify
the paracrine effects of VEGF in stimulating angiogenesis.

10 To determine if ECs expressed VEGF in vivo, in situ hybridization was performed

using a murine VEGF 1 65 cRNA probe to identify VEGF mRNA in the mouse ischemic
hindlimb (Coufifinhal T., et si.. Am J Pathol 1998;152:1667-1679). Prior to surgery,
scarce hybridization was detected in the ischemic limb. VEGF mRNA was also detected
in ECs located in small capillaries or venules. In some cases, ECs of larger caliber veins

15 also displayed positive hybridization for VEGF; VEGF expression among ECs of similar
caliber arteries, however, was less frequent.

Finally, we investigated the impact of hypoxia or hypoxia-dependent conditions
on VEGF receptor expression (Brogi E, et al., J Clin Invest 1996;97:469-476). HUVECs
and microvascular ECs (MVECs) were exposed to direct hypoxia or to medium

20 conditioned (CM) by myoblasts maintained in hypoxia for 4 days. Control ECs were
maintained in normoxia or normoxia-CM. Binding of *^^I-VEGF to ECs was then
evaluated. Hypoxic treatment of ECs had no effect on '^^I-VEGF binding. However,
treatment of ECs with hypoxia-CM produced a 3-fold increase in *^^I-VEGF binding,
with peak at 24 h (p<0.001, ANOVA). Scatchard analysis disclosed that increased

25 binding was due to a 13 -fold increase in KDR receptors/cell, with no change in KDR
affinity (Kd=260+51 pM, normoxia-CM versus Kd=281j:94 pM, hypoxia-CM) and no
change in EC number (35.6+5.9x10^ ECs/cm^ normoxia-CM versus 33.5+5.5x10^
ECs/cm^, hypoxia-CM). Similar results were obtained using CM from hypoxic SMCs.
KDR upregulation was not prevented by addition to the hypoxia-CM of neutralizing

30 antibodies against VEGF, tumor necrosis factor-a , transforming growth factor-bl or
bFGF. Similarly, addition of VEGF or lactic acid to the normoxia-CM had no effect on
VEGF binding. These experiments implicated a paracrine mechanism initiated by

wo 00/62798

-38-

PCT/USOO/09765

hypoxia that induces KDR receptor upregulation in ECs. Hypoxic cells, not only can
produce VEGF, but can also modulate its effects via paracrine induction of VEGF
receptors in ECs.

B. Hyperchol^terolemia attenuates the response to therapeutic
5 angiogenesis.

We investigated the anatomic extent and functional capacity of the collateral bed
which develops in response to limb ischemia in a well characterized animal model of
spontaneous hypercholesterolemia, the Watanabe heritable hyperlipidemic (WHHL)
rabbit (Van Belle E, et al., Circulation 1997;96:2667-2674\ We further characterized the

10 impact of exogenous angiogenic cytokine administration on collateral vessel development
and function in the same animal model. Weight-matched 6-month old male homozygous
WHHL {n=9) and normal NZW (n=9) rabbits underwent operative resection of one
femoral artery. Ten days later, the ischemic hindlimb was evaluated for collateral vessel
formation, blood flow, and tissue damage. Collateral vasculature was less extensive

15 among WHHL than NZW, as indicated by a significant reduction in angiographic score
(0.19±0.02 vs 0.35±0.03; P < .001) and capillary density (46.4±4.1 vs 78.9±4.6/mm^, P <
.0002). This was associated with a reduction in calf blood pressure index (9.5±3.5 vs
32.8 ± 2.8 %, P <.0001), arterial blood flow (7.5±0.6 vs 13.6db0.7 mL/min, P <.0001),
muscle perfusion index (40.1±3.2 vs 65.9±2.0%, P <.0001), and an increase in muscle

20 necrosis (48.16i5.41 vs 25.90±3.83% negative 2,3,5-triphenyltetrazolium chloride

staining, P<.004). Treatment of WHHL rabbits (n=9) with recombininant human VEGF
produced a statistically significant improvement in all functional as well as anatomic
indices of collateral development. Thus, collateral vessel development associated with
hindlimb ischemia in vivo is severely attenuated in an animal model of spontaneous

25 hypercholesterolemia, but may be nevertheless augmented by administration of
angiogenic cytokines.

These findings were confirmed in a murine model of hypercholesterolemia
(Couffinhal T, et al, Circulation 1999;(In Press)), the ApoE"'* mouse, with unilateral
hindlimb ischemia. Hindlimb blood flow and capillary density were markedly reduced in

30 ApoE"'' mice vs C57 controls. This was associated with reduced expression of VEGF in
the ischemic limbs of ApoE*'" mice. Cell-specific immimostaining localized VEGF
protein expression to skeletal myocytes and infiltrating T cells in the ischemic limbs of

wo 00/62798 PCT/USOO/09765

-39-

C57 mice; in contrast, T cell infiltrates in ischemic limbs of ApoE*^* mice were severely
reduced (despite normal absolute T cell covmts in these animals). The critical
contribution of T cells to VEGF expression and collateral vessel grov^ was reinforced
by the finding of accelerated limb necrosis in athymic nude mice with operatively

5 induced hindlimb ischemia. Adenoviral VEGF gene transfer to ApoE"^' mice resulted in
marked augmentation of hindlimb blood flow and capillary density. These findings thus
underscore the extent to which hyperlipidemia adversely affects native collateral
development, but does not preclude augmented collateral vessel growth in response to
exogenous cytokines. Moreover, results obtained in the ApoE"^' and athymic nude mice

10 imply a critical role for infiltrating T cells as a source of VEGF in neovascularization of
ischemic tissues.

C, The response to therapeutic angiogenesis is attenuated in a diabetic
animal model of hindlimb ischemia.

We determined if diabetes could: 1) impair the development of new collateral

15 vessel formation in response to tissue ischemia, and 2) inhibit cytokine-induced
therapeutic neovascularization (Rivard A, et al,, Am J Pathol 1999;154:355-364).
Hindlimb ischemia was created by femoral artery ligation in non-obese diabetic mice
(NOD mice, n=20) and in control C57 mice (n=20). Hindlimb perfijsion was evaluated by
serial laser Doppler studies after surgery. In NOD mice, measurement of the Doppler

20 flow ratio (DFR) between the ischemic and the normal limb indicated that restoration of
perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery,
DFR in the NOD mice was 0.49±0.04 vs. 0.73±0.06 for the C57 mice (p< 0.005). This
impairment in blood flow recovery persisted throughout the duration of the study, with
DFR values at day 35 of 0.50±0.05 vs 0.90±0.07 in the NOD and C57 mice respectively

25 (p < 0.001). CD3 1 immunostaining confirmed the laser Doppler data by showing a

significant reduction in capillary density in the NOD mice at 35 days after surgery (302±4
capillaries/nmi^ vs 782i78 in C57 mice (p < 0.005). The reduction in neovascularization
in the NOD mice was the result of a lower level of VEGF in the ischemic tissues, as
assessed by Northern blot, Westem blot and immunohistochemistry. The central role of

30 VEGF was confirmed by showing that normal levels of neovascularization (compared to
C57) could be achieved in NOD mice that had been supplemented for this growth factor
via IM injection of an adenoviral vector encoding for VEGF. We concluded that: 1)

wo 00/62798 PCTA:S00/09765

-40-

diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in
new blood vessel formation results from reduced expression of VEGF; and 3) cytokine
supplementation achieved by IM adeno-VEGF gene transfer restores neovascularization
in a mouse model of diabetes.

5 EXAMPLE 3 ; The impact of growth factor selection, mode of delivery, and use of
adjunctive therapies in optimizing the anatomic and physiologic outcomes of
therapeutic angiogenesis.

A. The character and magnitude of cellular proliferation observed in
response to VEGF may be modified by co-administration of a second angiogenic

10 growth factor.

To test this hypothesis, we evaluated the extent of neovascularization which was
achieved in vivo following intra-arterial administration of VEGF (500 |ig) alone, bFGF
(10 ^g) alone, and VEGF(500 ng)+bFGF(10^g) all as recombinant protein, to the
intemal iliac artery of the rabbit ischemic hindlimb (Asahara T, et al., Circulation

15 1995;92:II-365-II-37l\ Augmentation of calf blood pressure ratio as well as papaverine-
induced maximum flow reserve was significantly (p<0.05) greater in the VEGF+bFGF
group than the VEGF, bFGF, or saline control groups. The extent of neointimal
thickening in the intemal iliac artery at day 30 was not significantly different among the
foiu- experimental groups. This study thus demonstrated that co -administration of VEGF

20 and bFGF produces a greater (and parenthetically more rapid) improvement in vascularity
than either VEGF or bFGF alone.

We have also demonstrated that the pleiotropic effects of certain grov^ factors
may potentiate angiogenesis due to a combination of direct effects on EC proliferation
and migration, and indirect effects that result in the generation of other potent EC

25 mitogens from non-EC populations (Van Belle E, et al.. Circulation 1998;97:381-390).
In the case of hepatocyte growth factor (HGF), the synergistic effect, which results from
simultaneous administration of VEGF in vitro, is reproduced in vivo by HGF-induced
upregulation of VEGF in vascular SMCs. HGF is a pleiotropic growth factor, which
stimulates proliferation and migration of ECs via the c-Met receptor, present on ECs as

30 well as other cell types, including SMCs. We studied the effects of recombinant human
(rh) HGF in vitro, and in vivo in our rabbit model of hindlimb ischemia. We further
compared these effects with those of recombinant human VEGF (rhVEGFiss).

wo 00/62798

PCT/USOe/09765

-41-

In vitro, rhHGF and rhVEGFies exhibited similar effects on proliferation and
migration of ECs. When both cytokines were administered together, the result was an
additive effect on EC proliferation, and a synergistic effect on EC migration. Application
of rhHGF to cultures of human SMCs resulted in the induction of VEGF mRNA and

5 protein. In vivo, administration of rhHGF (500 [ig X 3) was associated with significant
improvements in collateral formation (p<.001) and regional blood flow (p<.0005), and
with a significant reduction in muscle atrophy (p<.0001). These effects were significantly
more pronoxmced than those of rhVEGFi65 administered according to the same protocol
(p<.05). Neither remote angiogenesis nor other pathologic sequellae were observed with

10 either rhHGF or rhVEGFi65. Thus, the finding of a potentiated angiogenic effect of
rhHGF via induction of VEGF constitutes what is in essence paracrine amplification of
angiogenesis

Finally, we tested the hypothesis that gene transfer of plasmid DNA encoding
angiopoietinl (Angl) and Ang2 could modulate collateral vessel development in a rabbit

15 model of hindlimb ischemia (Shyu K-G, et al.. Circulation 1998;98:2081-2087). Angl,
but not Ang2, gene transfer produced anatomic and physiologic evidence of enhanced
collateral vessel formation because Angl is known not to have any effect on EC
proliferation, it is possible that a synergistic effect between exogenous Angl and
endogenous VEGF expression accounts for the finding of enhanced collateral

20 development in response to Ang 1 .

B. The magnitude of angiogenesis developing in response to administration
of exogenous growth factors may be augmented by administration of heparin.

Ten days after excision of the femoral artery in one limb of NZW rabbits, heparin
(800 lU, n=13), VEGF (1 mg, n=3; 5 mg, n=5), heparin (800 lU) + VEGF (1 mg, n=5; 5

25 mg, n=7), or saline (n=8) was injected as a single bolus in a marginal ear vein (Banters C
et al., J Vase Surg 1 995 ;2 1 :3 14-325). Collateral vessel formation and limb perfiision were
assessed 10 and 30 days after treatment. Animals in both VEGF-treated groups had a
significantly higher (p <0.01) increase in calf blood pressure ratio at day 10 (control =
0.44±0.02; heparin = 0.47±0.02; VEGF = 0.60±0.01; [heparin + VEGF] = 0.61±0.02) and

30 day 30 (control = 0.49±0.05; heparin = 0.48±0.02; VEGF = 0.70db0.03; [heparin + VEGF]
= 0.73±0.03). Both VEGF-treated groups had a significantly higher (p <0.05)
angiographic score at day 30 (control = 0.28±0.01; heparin = 0.28±0.01; VEGF =

wo 00/62798 PCT/US00/0976S

-42-

0.37±0.01 ; [heparin + VEGF] = 0.38±0.02). Maximum flow reserve at day 30 in the
ischemic limb was higher (p <0.05) in VEGF-treated rabbits (control = 1 .87±0.07;
heparin = 1.92±0.08; VEGF = 2.42±0.16; [heparin + VEGF] = 2.33±0.12). Capillary
density was higher (p <0.01) in the ischemic muscles of VEGF-treated rabbits (control =
156±10/nim^ heparin = 178±8/mm^ VEGF = 230±10/mm^ [heparin + VEGF] =
233±8/nmi^). This series of in vivo experiments demonstrated that intravenous
administration of VEGF, vwth or without heparin, results in both anatomic and
physiologic evidence of enhanced collateral vessel formation in the rabbit ischemic
hindlimb.

C, Coincident activation of plasminogen facilitates therapeutic angiogenesis.

The requirement that ECs must remove certain constraining physical influences,
including the attachment to their underlying basement membrane and the more peripheral
barrier posed by their extracellular matrix, to facilitate cell movement for development of
a neovascular sprout, represents for angiogenesis a fundamental tenet (Vernon RB, et al..
Am J Pathol 1995;147:873-883). Houck et al (Houck KA, et al., J Biol Chem
1992;267:26031-26037) previously reported that cleavage by plasmin of VEGF189 at its -
COOH terminus generates a 34 kD proteolytic fi-agment (cl-VEGFigp) which is mitogenic
for ECs and active as a permeability agent. Park et al (Park JE, et al., Mol Biol Cell
1993;4:1317-1326) have reported that longer forms of VEGF are stably incorporated into
the extracellular matrix, but can become available in diffusible form when the matrix is
degraded by plasmin. We are currenUy investigating the possibility that coordinated
extracellular matrix degradation - achieved by generating plasmin via administration of
recombinant t-PA or co-transfection of plasmid cDNA encoding t-PA - may facilitate
therapeutic angiogenesis.

D. The magnitude of the angiogenic response does not vary as a function of
the distance from the ischemic site at which VEGF is administered.

The series of in vivo experiments described above (Example 3,B) established that
intravenous administration of VEGF, with or without heparin, results in both anatomic
and physiologic evidence of enhanced collateral vessel formation in the rabbit ischemic
hindlimb (Banters C, et al., J Vase Surg 1995;21 :3 14-325). A similar series of
experiments demonstrated that IM administration of VEGF recombinant protein
improved hindlimb perfusion to a similar extent as was seen vwth intra-arterial (and

wo 00/62798

^3-

PCT/USOO/09765

intravenous) delivery of the protein (Takeshita S, et al.. Circulation 1994;90:II-228-II-
234).

E. The magnitude of angiogenesis observed in response to VEGF varies as
a function of the isoform of VEGF employed.

5 Plasmid DNA encoding each of the three principal human VEGF 1 (VEGF A)

isoforms (phVEGFai, phVEGFi65, or phVEGFigg) was apphed to the hydrogel polymer
coating of an angioplasty balloon, and delivered percutaneously to one iliac artery of
rabbits with operatively induced hindlimb ischemia (Takeshita S, et al., Lab Invest
1996;75;487-502). Compared to control animals transfected with LacZ, site-specific

10 transfection of phVEGF resulted in augmented collateral vessel development documented
by serial angiography, improvement in calf blood pressure ratio (ischemic/normal limb),
resting and maximum blood flow, and capillary/myocyte ratio. Similar results were
obtained with phVEGF^i, phVEGFies, and phVEGFi89, suggesting that these isoforms
are biologically equivalent with respect to in vivo angiogenesis. The fact that viral or

15 other adjunctive vectors were not required further suggests that secreted gene products
may have potential therapeutic utility even when the number of successfully transfected
cells remains low. Arterial gene transfer of naked DNA encoding for a secreted
angiogenic cytokine thus represents a potential alternative to recombinant protein
administration for stimulating collateral vessel development.

20 F. Therapeutic angiogenesis may be effectively performed using direct

intramuscular (IM) administration of the gene encoding VEGF.

Striated muscle had been shown to be capable of taking up and expressing foreign
genes transferred in the form of naked plasmid DNA, though typically with a low level of
gene expression. We had shown, however, that in the case of genes which encode

25 secreted proteins, low transfection efficiency did not preclude bioactivity of the secreted
gene product (Takeshita S, et al.. Lab Invest 1994;71:387-391) (Losordo DW, et al.,
Circulation 1994;89:785-792). Accordingly, we investigated the hypothesis that IM gene
therapy with naked plasmid DNA encoding VEGF could augment collateral development
and tissue perfusion in the rabbit ischemic hindlimb (Tsurumi Y, et al.. Circulation

30 1996;94:3281-3290).

Ten days after ischemia was induced in one rabbit hindlimb, 500 \ig of
phVEGFi65, or the reporter gene LacZy were injected IM into the ischemic hindlimb

wo 00/62798

-44-

PCTAJSOO/0976S

muscles. Thirty days later, angiographically recognizable collateral vessels and
histologically identifiable capillaries were increased in VEGF-transfectants compared to
controls. This augmented vascularity improved perfusion to the ischemic limb,
documented by a superior calf blood pressure ratio for phVEGFiss (0.85i0.05) vs.

5 controls (0.64±0.05, p<.01); improved blood flow in the ischemic limb (measured using
an intra-arterial Doppler wire) at rest (phVEGFi65=21.3±3.9, control=14.6±1.6 ml/min,
p<.01) and following a vasodilator (phVEGFi65=54.2±12.0, control=37.3±8.9, p<.01);
and increased microspheres in the adductor (phVEGFi65=4.3i:1.6, control=2.9±1.2
ml/min/lOOg tissue, p<.05), and gastrocnemius (phVEGFi65=3.9dbl.O, control=2.8±1.4

10 ml/min/ lOOg tissue, p<.05) muscles of the ischemic limb. These experiments established
that ischemic skeletal muscle constituted a promising target for gene therapy with naked
plasmid DNA. IM transfection of genes encoding angiogenic cytokines, particularly
those which are naturally secreted by intact cells, thus appeared to represent an alternative
treatment strategy for patients in whom extensive vascular disease prohibited access to

15 the lower extremity vasculature otherwise required for intra-arterial catheter-based gene
transfer.

Based on this animal data, we initiated a phase I clinical trial to (1) document the
safety and feasibility of IM gene transfer using naked plasmid DNA encoding VEGF, and
(2) analyze potential therapeutic benefits in patients with CLI (Baumgartner I, et al.,

20 Circulation 1998;97:1 1 14-1 123) (Isner JM, et al., J Vase Surg 1998;28:964-975).

Gene transfer was performed in 1 0 limbs of 9 patients with non-healing ischemic
ulcers (n=7/10) and/or rest pain (n=10/10) due to peripheral arterial disease. A total dose
of 4000 ^ig of phVEGFi65 was injected directly into the muscles of the ischemic limb.
Gene expression was documented by a transient increase in serum levels of VEGF

25 monitored by ELISA assay. The ankle-brachial index improved significantly (0.33 ±
0.05 to 0.48 ± 0.03, p=0,02), new collateral blood vessels were directly visualized by
contrast angiography in 7 limbs, and magnetic resonance angiography showed qualitative
evidence of improved distal flow in 8 limbs. Ischemic ulcers healed or markedly
improved m 4/7 limbs, including successful limb salvage in 3 patients recommended for

30 below-knee amputation. Tissue specimens obtained from an amputee 10 wks after gene
therapy showed foci of proliferating ECs by immunohistochemistry. PGR and Southern
blot analyses indicated persistence of small amounts of plasmid DNA. Complications

wo 00/62798

PCTAJS00/0976S

-45-

were limited to transient lower extremity edema in 6 patients, consistent with VEGF-
enhancement of vascular permeability.

These findings thus demonstrated that IM injection of naked plasmid DNA may
achieve constitutive overexpression of VEGF sufficient to induce therapeutic
5 angiogenesis in selected patients with CLI.

Most recently, we adapted the use of IM phVEGFies gene transfer to investigate
gene therapy for therapeutic angiogenesis in patients with myocardial ischemia (Losordo
DW, et al.. Circulation 1998;98:2800-2804). A phase 1 clinical study was initiated to
determine the safety and bioactivity of direct intramyocardial gene transfer of VEGF as
10 sole therapy for patients with symptomatic myocardial ischemia.

VEGF gene transfer (GTx) was performed in 5 patients (all male, ages 53-71)
with angina due to angiographically documented coronary artery disease (CAD) who had
failed conventional therapy (drugs, angioplasty surgery). Naked plasmid DNA encoding
VEGF (phVEGFi65) was injected directly into the ischemic myocardiimi via a "mini" left
15 anterior thoracotomy. Injections caused no changes in heart rate (pre-GTx=75±15/min vs
post-GTx=80±16/min, p=NS), systolic BP (1 14±7 mmHg vs 118±7mm Hg, p=NS), or
diastolic BP (57±2 mmHg vs 59±2 nrmiHg, p=NS). Ventricular arrhythmias were limited
to single unifocal premature beats at the moment of injection. Serial ECGs showed no
evidence of new myocardial infarction in any pt. Intraoperative blood loss was 0-50cc
20 and total chest tube drainage was 1 10-395cc. Cardiac output fell transiently post-op but
increased within 24 hrs (pre-anesthesia = 4.8±0.4 vs post-anesthesia = 4.1±0.3 vs 24 hrs
post-operatively = 6.3±0.8, p=0.02). Time to extubation following closure was 18.4±1 .4
min and avg post-op hospital stay was 3.8 days. All patients had significant reduction in
angina (NTG use = 53.9+10.0/wk pre-GTx vs 9.8±6.9/wk post-GTx, p<0.03). Post-
25 operative left ventricular ejection fraction (LVEF) was either unchanged (n=3) or

improved (n=2, mean increase in LVEF = 5%). Objective evidence of reduced ischemia
was documented using dobutamine SPECT-sestamibi imaging in all patients. Coronary
angiography showed improved Rentrop score in 5/5 patients.

This initial experience with naked gene transfer as sole therapy for myocardial
30 ischemia suggested that direct intramyocardial injection of naked plasmid DNA via a
minimally invasive chest wall incision is safe and may lead to reduced symptoms and
improved myocardial perfusion in selected patients with chronic myocardial ischemia. As

wo 00/62798

^6-

PCT/USOO/09765

of February, 1999, a total of 23 patients have been treated in this fashion with similar
results (Symes JF, et al., Ann Thorac Surg 1999).

In summary, the above-described experiments extended the notion of therapeutic
angiogenesis from in vitro studies and animal models to patients with lower extremity
5 and myocardial ischemia.

EXAMPLE 4 : Peripheral Neuropathy

!• Neurological findings in patients undergoing phVEGFi^sgene transfer.

Methods and results: We prospectively evaluated neurological and
neurophysiological findings in patients undergoing phVEGFies gene transfer for critical

10 limb ischemia (CLI). All patients were evaluated by two neurologists, one performing
clinical assessment, and one performing electrophysiologic testing. Both were blinded to
each other's results, and both were blinded to the results of patients' vascular
examinations. Furthermore, at follow-up examinations, both were blinded to results of
previous examinations. Quantitative sensory testing was performed using CASE IV

15 (Computer Aided Sensory Evaluator, a quantitative sensory testing device for thermal
pain and vibration thresholds (Dyck PJ, et al., Diabetes Care 1987;10:432-440)). Tibial
motor, peroneal motor, and sural sensory electrophysiologic studies were performed
using standard techniques. Both lower extremities were studied, except in patients in
whom the contralateral, non-treated limb was not available due to previous amputation. A

20 total of 24 limbs have thus far been analyzed before and 3 mo after gene transfer; 19 of
these have been followed to 6 months.

These findings suggest that therapeutic angiogenesis have a favorable impact on
established ischemic peripheral neiiropathy. With regard to symptoms, for example, as
early as 3 mo. Symptom Score, encompassing 5 neuropathy-related symptoms, decreased

25 (i.e. improved) from (m=bSEM) 3.3±0.5 to 1.7±0.4 (p<0.01). Sensory Disability Score
decreased from 9.5±1 .3 to 7.4±1.2 (p<0.01). By 6 mo. Symptom Score decreased to
1 .1±0.4 (p<0.01 vs baseline of 3.4). Sensory Disability Score likewise decreased to
6.3±1.4 (p=0.01 vs baseline 9.5±1.3), along with a reduction in Total Disability Score
(12.7±2.1 to 9.2±1.7, p=0.01). Vibration threshold decreased (i.e. improved) fi:om

30 21 .0±0.9 to 19.8il .0, p=0,04. Moreover, by 6 months, improvement in objective indices
of nerve function became manifest. Peroneal motor nerve amplitude increased from

wo 00/62798

-47-

PCT/USOO/09765

2. liO.6 to 2.8i:0.6 (p=0.03). In no case did clinical examination or electrodiagnostic
studies show statistically significant improvement for measurements
recorded from the contralateral, non-treated lower extremity. The percent change in
peroneal motor amplitude in the treated vs untreated leg for each of the 19 patients
5 followed for 6 months (one patient, who began with an amplitude of 0 and increased to
0.2 is not shown); illustrates that increases were observed in three insulin-requiring
diabetics (patients 1,5, and 11). Also included was one patient in whom we documented
the appearance of a previously absent potential. For the group as a whole, the percent
increase in peroneal amplitude (50.3±21.0) exceeded the change observed in the non-

10 treated leg (-10.8^7.9) to a statistically significant degree (p=0.02). Similarly, the simi of
motor amplitude increased 16.5±8.7% vs the non-treated leg (-8.8±6.5), (p=0.04).

Conclusions: This prospective study - the first to our knowledge to investigate
the impact of therapeutic angiogenesis on peripheral nerve function in patients with limb
ischemia - showed evidence of improvement in peripheral nerve function in patients

1 5 undergoing phVEGF i es gene transfer.

2. Adaptation of the rabbit ischemic hindlimb model for the investigation of
ischemic peripheral neuropathy.

Methods and results: The rabbit model of hindlimb ischemia has been
extensively characterized in our laboratory and frequently utilized to investigate strategies

20 of therapeutic angiogenesis. We therefore xmdertook a series of preliminary experiments
to determine the extent of peripheral neuropathy which might accompany the
development of vascular insufficiency in this model.

Determination of normal electrophysiologic parameters. All protocols were
approved by the Institutional Animal Care and Use Committee at St. Elizabeth's Medical

25 Center. Male NZW rabbits 5-6 yrs of age (mean weight = 5 kg) were used for all

experiments. Pilot experiments performed in our laboratory showed that when hindlimb
ischemia is created in young (6-8 mo) NZW rabbits, development of neuropathy is
transient and of unpredictable magnitude. We therefore performed pilot experiments in
old (5 yrs) rabbits, in which angiogenesis was shown to be retarded (Rivard A, et al.,

30 Circulation 1 999;99:3 11-1 20) and documented persistent and profound neurophysiologic
abnormalities. Before recordings from ischemic limbs could be made, it was necessary to
document the electrophysiologic responses recorded from the rabbits' non-ischemic

wo 00/62798 PCT/US00/0976S

-48-

hindlimb. Motor and sensory nerve potentials were recorded from both limbs of 8 rabbits
with intact femoral arteries. Data were considered as mean ± standard error of the
(m±SEM).Compound muscle action potentials (CMAPs) were 16.0±1.2 mVin the left
limb and 16.4±0.9 mV in the right limb (p=ns). Sensory nerve action potentials (SNAPs)
5 were 7.7±0.8 ^V in the left limb vs 8. 1±1 . 1 jiV in the right limb (p=ns).

(Neurophysiologic recordings in the non-ischemic limbs of old rabbits were not different
from those recorded in non-ischemic limbs of yoimg rabbits).

Induction of ischemia in the rabbit hindlimb. Unilateral hindlimb vascular
insufficiency was operatively created in 10 rabbits using previously published techniques

1 0 (Takeshita S, et al., J Clin Invest 1 994;93 :662-670).

Verification of ischemic peripheral neuropathy. After surgical induction of
unilateral hindlimb ischemia, rabbits were allowed to recover for 5 days.
Electrophysiologic recording of peripheral nerve function in the ischemic and non-
ischemic limbs was then performed xmder general anesthesia on a weekly basis for 3

15 months CMAPs and SNAPs were recorded. Motor nerve conduction velocities (MCV)
and sensory nerve conduction velocities (SCV) were calculated. Concomitantly, the calf
blood pressure ratio, defined as the ratio of systolic pressure of the ischemic limb to that
of the normal limb, was determined for each rabbit using standard techniques. At the
defined end point, 12 wks after induction of ischemia, rabbits also underwent selective

20 angiography of the internal iliac artery as previously described (Takeshita S, et ah, 7 C/m
/rtve5M994;93:662-670).

The development of hindlimb ischemia had a profound effect on peripheral nerve
function. Data were considered to represent function in right (ischemic) limb as a
percentage of that recorded in left (non-ischemic) limb. Motor nerve amplitudes,

25 indicated by CMAP, dropped to zero post-operatively before they became detectable

again 4 wks later; at 8 wks post-operatively, CMAPs were still only 10 to 15% of normal.
From wk 8 on, CMAPs increased in a nearly linear fashion, improving to 50-60% of
normal by wk 10, vwth no further changes through wk 12 (56.2±7.3%). MCV behaved
similarly: ischemic limbs displayed non-determinable conduction velocities up to wk 3.

30 From wk 4 on, MCV increased in a nearly linear pattern, peaking at 87.4±3.6% of normal
by wk 12.

wo 00/62798 PCT/USOO/09765

-49-

In contrast to motor nerve parameters, SNAPs did not drop to zero level following
induction of ischemia, but did remain depressed at 1 7 - 22% of normal between wks 1 to
4 following induction of ischemia. From then on, SNAPs increased to 51.7±5.1% of
normal at wk 12. In contrast to MCV and CMAP, which were undetectable after the

5 induction of ischemia, SCVs were initially reduced to approximately 20%, before they
started to rise at wk 3 and by wk 12 were 98.9±0. 1% of normal. Ischemia reduced calf
blood pressure ratio to 22.9±5.4% at wk 1 . An almost linear increase in calf blood
pressure ratio peaked at 79.0±9.0% at wk 12. Finally, morphometric angiographic
analysis, performed at wk 12, revealed that the angiographic score (quantitative analysis

1 0 of vascular density based on contrast angiograms for ischemic limbs was 44.8±4.5% of
non-ischemic limbs, consistent with endogenous neovascularization described previously
for this model (Takeshita S, et al., J Clin Invest 1994;93:662-670). Pathologic findings
included focal loss of axons and disrupted myelin sheaths.

Conclusion: These findings indicate that hindlimb ischemia causes a severe

15 peripheral neuropathy which affects both motor and sensory nerve functions. The rabbit
model of hindlimb ischemia thus represents a suitable tool for the investigation of
ischemic peripheral neuropathy.

3. Therapeutic angiogenesis attenuates the development of ischemic
peripheral neuropathy.

Methods and results. Previous work from our laboratory has demonstrated that
IM phVEGFi65 gene transfer performed at the time of siugery to create unilateral

25 hindlimb ischemia results in accelerated revascularization of the ischemic hindlimb, in
comparison to control animals injected with a reporter gene (Tsurumi Y, et al.,
Circulation 1996;94;328 1-3290). Accordingly, we performed a pilot study involving 10
rabbits, each of which received 5 injections of phVEGFi65 (100 fig @) into the adductor
(2 sites), medial large (2 sites), and semimembranosus (one site) muscles at the time the

30 animals imderwent unilateral excision of the femoral artery to create hindlimb ischemia.
Following surgery and gene transfer, rabbits underwent electrophysiologic and vascular
examinations on a weekly basis. Immediately prior to sacrifice at 12 wks, selective

wo 00/62798 PCTAJSOO/05765

-50-

angiography was performed. Postmortem examination included analysis of
capiliary/myocyte ratios and morphometric examination of nerve tissue sections.

One week post-surgery, CMAPs recorded in the ischemic limb were reduced to
36.8±9.1% of that recorded from the left limb. At wk two, however, CMAPs had
5 improved to 49.9±0.5% of normal, and by 3 wks improved further to 82.0i:3.4% of
normal. CMAP ratios for wks 8 to 12 displayed a plateau-like pattern with values
ranging from 83.9dbl .3% at wk 8 to 90,5±4.3% at wk 12. In comparison, CMAPs for the
controls were 10.9±2.9% of normal at wk 8, and 56.2±7.3% at wk 12. From wk 8 to wk
12, CMAPs were significantly higher at each timepoint in day 0-treated rabbits.

10 Measurements of MCV demonstrated a moderate drop in conduction velocity for

a brief period of two wks after the induction of ischemia (83. 5^1 1 .0% and 89.9±7.1% at
wks 2 and 3, respectively). MCVs at these 2 timepoints were significantly different from
values in the non-ischemic limb. Thereafter, however, MCV recovered to normal values,
from wk 4 through wk 12.

15 Examination of sensory nerve ftmction disclosed a reduction in SNAPs from

100% at baseline to 70.2±1 1.3% within the first wk after surgery. This parameter
continued to be significantly lower than normal up to wk 4. Neurophysiological
assessment at wk 5, however, disclosed improvement in SNAPs to 99.6dbl6.6%, hence no
longer significantly different from normal baseline values. No fiirther significant change

20 in SNAPs was observed up to wk 12 when the animals were sacrificed.

SCVs were also recorded in this study group and were foimd to be only slightly
depressed within the first wks after the onset of ischemia. The lowest SVC was
90.6±7.3% at wk 3. All other timepoints were even closer to normal or absolutely normal,
and none of the values taken differed significantly from normal vdues.

25 At each timepoint employed for neurophysiological examinations, blood pressure

ratios for the rabbits' hindlimbs were recorded as well. The results with the motor nerve
parameters show that post-operatively there was an expected drop in the blood pressure
ratio to 38.0±9.1% of normal. Three wks post-operatively, the calf blood pressure ratio
had risen again to 82.9±4.7%. Further follow-up examinations showed a progressive rise

30 in blood pressure ratio to 97.2±1 . 1% at wk 12 before sacrifice. Pathologic observations
indicated improved preservation of axons and myelin sheaths, compared to imtreated
animals.

wo 00/62798 PCTAJSCO/09765

-51-

Conclusion: Day-0 IM gene transfer of phVEGFi6s prevented CMAPs as well as
SNAPS from dropping to unrecordably low levels, and led to significantly faster recovery
of CMAPs and SNAPs. An even more obvious impact of treatment was seen on MCV
and SCV, both of which were only mildly reduced within the first 3 wks after ischemia.
5 Improvement in calf blood pressure ratio paralleled these neurophysiologic findings.

4. Therapeutic angiogenesis promotes recovery of established ischemic
peripheral neuropathy.

Methods and results: To investigate whether established ischemic neuropathy can
be reversed by therapeutic angiogenesis, 10 rabbits underwent excision of the femoral
10 artery followed by a 10 day interval to permit development of severe hindlimb ischemia
and hence ischemic neuropathy. At day 10, rabbits underwent phVEGFies gene transfer
for therapeutic angiogenesis as outlined above. Neuroelectrophysiologic and blood
pressure measurements were again performed on a weekly basis according to the protocol
described above.

15 Within the 10-day period between induction of ischemia and treatment, CMAPs

dropped to undetectable levels. At week 4 (2.5 wks post-treatment), amplitudes were
16.8±1.6% of baseline. Subsequent follow-up examinations documented improvement in
CMAPs, increasing to 44. ld:4.7% at wk 6, and 69. 1±2.2% at the endpoint (wk 12 after
the induction of ischemia, i.e. 10.5 wks post-gene transfer).

20 MCVs were detectable neither at first examination after excision of the femoral

artery, nor briefly after gene therapy. However, by wk 3 (1 .5 wks after gene transfer),
MCV increased to 35.0±14.1% of normal, and one wk later to 74.8±3.2% of normal. By
wk 12, MCV in the ischemic limb was restored to 86.5±3.9% of normal.

SNAPs decreased to 38.3 ± 15.2% of normal following induction of ischemia.

25 The first significant change in SNAPs thereafter was detectable 2.5 wks after gene
therapy (wk 4), when the SNAP ratio improved to 64.9±8.4%. By wk 6, SNAPs had
reached normal values again (103.3±10.5%), and at wk 12 were 96.9±4.9%.

SCVs, in contrast to the MCVs, were less severely affected by induction of
ischemia: no significant alteration (in comparison to the contralateral non-ischemic

30 contralateral limb) could be detected.

Blood pressure measurements in this study group disclosed the anticipated
reduction in limb perftision post-surgery (blood pressure ratio in the ischemic to normal

wo 00/62798 PCTA;S00/09765

-52-

limb = 21.8±5.4%). Following gene therapy, blood pressure ratio showed an almost
linear increase, peaking at 95.2 ±0.3% by wk 12.

Conclusion: For SNAPs, the day O-treated group showed the most favorable
outcome in terms of minimally impaired nerve function and quick recovery. In the day
5 1 O-treated group, however, SNAPs were also restored earlier, reaching normal function
again by 4.5 wks after gene therapy. SNAPS in untreated animals remain reduced
(51.7±5.1% of normal values). CMAP recovery of day O-treated animals was fastest and
most complete, whereas onset of recovery in xmtreated rabbits was delayed to 6.5 wks
after surgery. CMAPs in the day 10-treatment group recovered faster than those in

10 untreated rabbits. These findings suggest a favorable effect of therapeutic angiogenesis on
the development of ischemic peripheral neuropathy.

5. Expression of VEGF and VEGF receptor by Schwann cells in vitro
Methods and results: NZW rabbits were sacrificed under aseptic conditions using
an intracardiac injection of pentobarbital sodium, following which their sciatic nerves

15 were dissected free, harvested, and processed as previously described (Morrissey TK, et
al., JNeurosci 1991 ;1 1 :2433-2442). Briefly, the nerves were placed into Liebovitz's L-15
medium (GIBCO) supplemented with 50U/ml penicillin and 0.05 mg/ml streptomycin (L-
15a). The epineurium, connective tissue, and blood vessels were stripped off with fine
forceps. The nerves were placed in fresh L-15a and minced with scissors or scalpel blades

20 into roughly 1x1 mm^ explants. Minced nerves were placed in a 35-mm dish containing
1.25 U/ml dispase (Boehringer Mannheim Biochemicals), 0.05% collagenase
(Worthington Biochemicals), 20% fetal bovine serum (FBS), and 25 mM HEPES in
Dulbecco's Modified Eagle's Mediiun (DMEM,GIBCO). The tissue in this solution was
triturated in a Pasteur pipette approximately 10 times and was then incubated overnight at

25 37**C in a humidified atmosphere of air with 5% CO2. The following day, the explants
were dissociated by gentle trituration through a flame-narrowed borosilicate pipette (0.5 -
1-mm bore), washed 2-3 times in DMEM/10% FCS, and seeded into poly-D-lysine-
coated (Sigma) tissue culture flasks (FALCON). To amplify the Schwann cell cultures,
the cells were grown on poly-D-lysine-coated tissue culture plastic in DMEM

30 supplemented with 10% FBS, 2 ^M forskolin (Sigma) and 10 ^g/ml bovine pituitary
extract (GIBCO).

wo 00/62798 PCT/US00/0976S

.53-

Schwann cells were identified in cultures on the basis of cell soma and nuclear
morphology lising phase microscopy. Cells with long hi- or tripolar processes (that were
approximately 5 -7 times the width of the cell body) and oval nuclei were counted as
Schwann ceils. In fixed cultures, we performed immunocytochemical labeling for SI 00
5 protein. Cells were fixed for 10 min in 4% paraformaldehyde followed by

permeabilization in 4% paraformaldehyde with 0.02% Triton X-100. After blocking with
L- 15/10% heat-inactivated horse serum, the cells were incubated with a mouse
monoclonal anti-SlOO (b-subunit) antibody (1:1000; Sigma) overnight at 4°C. The
following day, the cells were fiirther processed by incubation with a biotinylated

10 secondary horse anti-mouse antibody for 30 min at room temperature. Slides were then
labeled with an Ultra Streptavidin Enzyme Complex (Signet Laboratories) according to
the manufacturer's directions.

Schwann cell expression of VEGF and Flt-1 (VEGF receptor 1, or VEGFR-1)
protein expression was assessed by Western blotting. Cells were lysed by addition of 1

1 5 ml RIPA buffer (1% NP-40, 0.5% sodium deoxycholic acid, 0. 1 % SDS in PBS, pH 7.4; 1
^iM leupeptin; 5 \iM aprotinin; 1 mM PMSA; and 1 jiM pepstatin, all Sigma) per 100-
mm plate. Protein extracts (100 ng) were separated on a 10% SDS-PAGE and transferred
to a 0.2-fim PVDF membrane (Bio Rad). The membranes were blocked in 10% nonfat
dry milk/0.2% Tween-20 in PBS, pH 7.4, then immunoblotted with a mouse monoclonal

20 anti-human VEGF antibody (1 :250) or a mouse monoclonal anti-human Flt-1 antibody
(1 :500) (both antibodies firom Sigma) overnight at 4°C. Blots were washed with 0.2%
Tween-20 in PBS and incubated with horseradish peroxidase-linked goat anti-mouse
antibody (1:10000; Sigma) for 45 min. Imunoreactive bands were visualized with ECL
reagent (Amersham). Very recent data from our laboratory has identified mRNA for both

25 VEGFR2 (KDR) and neuropilin-1 in cultured Schwann cells as well.

We also assessed Schwann cell VEGF expression by ELISA testing upon the
culture mediimi of equal numbers of cells in 24-well tissue culture plates. After the cells
had grown to confluence, they were washed with DPBS and then further kept in DMEM
containing various concentrations of cytokine- and growth factor-fi'ee defined FBS. After

30 24 hrs of incubation, equal voliunes of supematants from each test condition were

removed, and the samples were cleared firom cell debris by centrifiigation (12000 rpm for
5 min). VEGF protein was determined with an immunoassay according to the

wo 00/62798 PCT/USOO/09765

-54-

manufacturer's instructions (R&D Systems). Results were compared with a standard
curve of human VEGF with a lower detection limit of 5 pg/ml. Samples were checked by
serial dilution and were performed in duplicate. The results are shown in Example Table
1.

5 Example Table 1. ELISA measurement of VEGF protein (n=2) for each condition

Defined FBS content in media

0.5% 1% 5% 10%

• VEGFi65 (pg/nal)

Control
medium

12.6

9.2

1.1

63.4

Cell culture
supernatant

851.5

759.8

1339.4

1935.5

Conclusion: Schwann cells express VEGF protein as well as VEGFR-1 (Flt-1) in

vitro.

6. Schwann cell migration in response to VEGF

10 Methods and results: The migratory response of both primary and subcultured

rabbit Schwann cells was assessed using a modified Boyden chamber assay (McCarthy
JB, et al., J Cell Biol 1983;97:772-777) (Baron-Van Evercooren A, et al., J Cell Biol
1982;93:21 1-216). Schwann cells were detached from the tissue culture flasks with
0.05% trypsin/EDTA (GIBCO) and resuspended at 3xlO^/ml in chemotaxis medium

15 (DMEM w/ 0.5% BSA, Sigma) before being placed in the upper well of a 48-welI
chemotaxis chamber (Neuroprobe). The lower wells of the chemotaxis chamber
contained test reagents, which were reconstituted in chemotaxis medium. Upper and
lower wells were separated by a 8-^m pore polyvinylpyrrolidone-free polycarbonate filter
(Poretics), precoated with fibronectin (20 ng/ml). Chemotaxis chambers were incubated

20 for 4 h at 37°C in a humidified atmosphere of air with 5% CO2. Thereafter, the filters
were fixed and stained with Diff Qick (DADE). The filters were cut in half and mounted
with the bottom side down (containing migrated cells) onto glass coverslips. Cells that
had not migrated were removed from the upper surface with cotton swabs. After air
drying, the coverslips were mounted onto glass slides and migration was quantified by

25 counting migrated cells in 20 randomly selected high power (X400) fields. Each sample
was assessed in quadruplicate, and results expressed as m±SEM chemotaxis index (CI,
the ratio between the number of cells that migrated toward test substances and those

wo 00/62798

PCT/USOO/09765

-55-

which migrated toward medium control; migration toward medium reflects spontaneous
migration). Zigmond-Hirsch checkerboard analysis (Zigmond SH, et al., J£xp Med
1973;137:387-410) was performed by incubating the cells in the chamber with various
doses of test substances either on the bottom of the filter (to establish a positive gradient),
5 on top of the filter (to establish a reversed gradient), or on both sides of the filter in equal
concentrations (to examine accelerated random movement). Statistical significance of
data was determined by Mann- Whitney U-test and Kruskal- Wallis-test for nonparametric
analysis of results.

The chemotactic response of Schwann cells to graded concentrations of

10 rhVEGFi65 protein was considered. VEGF stimulated migration in a dose-dependent
manner, with maximal activity ranging firom 10 to 1000 ng/ml. At these concentrations,
approximately 1.8 times more cells migrated through the polycarbonate filter in
comparison to the media control. At the lowest concentration tested (1 ng/ml),
rhVEGFi65 still induced a statistically significant increase in chemotaxis uith a CI of

15 1.3±0.04. Nerve growth factor (NOP, Sigma) served as positive chemoattractant control
(Anton ES,etal.,Proci\ra//^ca^ 5c/ 1/&4 1994;91:2795-2799). To check for the
specificity of this VEGF-induced effect, we performed additional experiments using a
neutralizing monoclonal anti-VEGF antibody (Sigma). Results demonstrate that the
antibody (dilution 1:500) itself had no effect on Schwann cell migration. When the

20 antibody was present together with rhVEGFies, we observed complete abrogation of the
stimulatory effect of VEGF at 100 ng/ml as well as at 500 ng/ml were completely
abrogated.

The migratory response of Schwann cells to VEGF could be explained either by
chemotaxis (directed movement of cells along a chemotactic gradient) or by

25 chemokinesis (enhanced speed or fi-equency of random migration). To address this issue,
we performed a series of checkerboard analyses (Example Table 2). Addition of VEGF
exclusively to the upper compartment together with Schwann cells failed to enhance
migration; in contrast, gradually increasing the concentration gradient of VEGF between
the lower and upper compartment increased migration of Schwann cells toward the lower

30 compartment.

Example Table 2 Checkerboard Analysis of rh VEGFi6.s-induced Schwann
Cell Migration.

wo 00/62798 PCTAJSOO/09765

-56-

VEGF (ng/ml), Upper compartment

100

500

VEGF(^g/ml)

15.93 ±1.1

15.05 ±0.9

14.03 ±0.9

Lower

16.40 ± 1.2

17.27 ±0.8

17.02 ± 1.4

16.43 ± 1.1

Compartment

100

27.45 ± 0.8

24.87 ±1.4

16.81 ± 1.1

17.93 ±0.7

500

33.90 ±1.2

33.12 ±2.2

27.56 ±1.9

15.56 ± 1.6

Conclusion: VEGF directly promotes Schwann cell chemotaxis.

It should be understood that the preceding is merely a detailed description of
certain preferred embodiments. It therefore should be apparent to those skilled in the art
5 that various modifications and equivalents can be made without departing from the spirit
and scope of the invention. The invention is intended to encompass all such
modifications within the scope of the appended claims.
EXAMPLE 5 :

Methods and results: Diabetes was induced in female Sprague Dawley rats by i.p.

10 streptozotocin (85 mg/kg) according to previously published methods. Blood glucose
was monitored repeatedly and animals were deemed diabetic if blood glucose exceeded
200 mg/dL. After a period of twelve weeks, the animals were treated with i.m. injections
of either 250 micrograms of make plasmid DNA encoding for human VEGFies, or saline.
The effects of treatment were assessed after four weeks by recording sciatic nerve motor

15 and sensory conduction velocities (M. Kalichman et al., Brain Res. 1998, 810:130-137).
The motor nerve conduction velocity (MCV) of the untreated, non-diabetic group was
48.8 ± 3.0 m/s (mean ± SEM), and the sensory nerve conduction velocity (SCV) of this
group was 62.0 ±4.9 m/s. The MCV of the saline-injected, diabetic group was 33,7 ±1 .3
m/s and the SCV was 27.1 ± 2.2 m/s. Therefore, MCV as well as SCV were significantly

20 slowed in the diabetic (saline-injected) rats, as compared to the non-diabetic control

animals. However, when rats were injected with ph VEGFies, MCV was restored to 41.9
±1.9 m/s, and SCV was improved to 59.5 ±7.0 m/s. Both MCV and SCV of the diabetic
VEGF-treated group are significantly different from the corresponding parameters of the
non-diabetic rats. Moreover, several non-diabetic rats were treated with ph VEGF) 65 as a

25 control, and neither MCV nor SCV were affected by this treatment.

Apfel et al. (Brain Res. 1994, 634:7-12) had reported that nerve growth factor
(NGF) could ameliorate a sensory neuropathy caused by diabetes in streptozotocin-

wo 00/62798 PCTA;S00/09765

-57-

treated rats. These investigators adrmnistered recombinant human NGF three times
weekly at a dose of 3 mg/kg. After 1 1 weeks, the compoimd latency as a measure of the
conduction velocity of the caudal nerve in the tail was measured. The imtreated diabetic
rats had a mean compoimd latency of 1 .85 ± .09 m/s, while the NGF-treated diabetic rats

5 had a mean compound latency of 1 .76 ±.04 m/s, and the control rats had a mean

compound latency of 1 .40 ±0.03. The rats from the treated group exhibited better nerve
function compared to the untreated group but their NCVs were still significantly lower
than those of the control group. We found that i.m. ph VEGFies was able to completely
restore both motor and sensory NCVs within four weeks.

10 References:

Kalichman MW, Dines KC, Bobik M, Mizisin AP, Nerve conduction velocity,
laser doppler flow, and axonal caliber in galactose and streptozotocin diabetes, Brain Res,
1998,810:130-137,

Apfel SC, Arezzo JC, Brownlee M, Federoff H, Kessler JA, Nerve growth factor
15 administration protects against experimental diabetic sensory neuropathy.

All references, patents and patent publications that are recited in this application
are incorporated in their entirety herein by reference.
What is claimed.

wo 00/62798 PCTAJSOO/09765

-58-

CLAIMS

1 . A method for treating a condition characterized by peripheral neuropathy
in a subject comprising:

administering at least one angiogenic growth factor to a subject in need of
5 such treatment in an amount effective to alleviate a symptom of peripheral neuropathy in
the subject,

wherein the subject is otherwise free of symptoms calling for treatment
with an angiogenic growth factor.

2. A method for treating a condition characterized by peripheral neuropathy
10 in a subject comprising:

administering at least one angiogenic growth factor to a subject in need of
such treatment in an amoxmt and for a period of time effective to alleviate a symptom of
peripheral neuropathy in the subject,

wherein the period of time is significantly greater than the period of time
15 during which an angiogenic growth factor is administered to a subject for the purpose of
revascularization of an ischemic tissue.

3. A method for treating a condition characterized by peripheral neuropathy
in a subject comprising:

administering at least one angiogenic growth factor to a subject in need of
20 such treatment in an amount effective to alleviate a symptom of peripheral neuropathy in
the subject,

wherein administering is by intramuscular injection into a tissue at an
injection site that is proximate to a nerve suspected of a neuropathy and distal to an
injection site that would be selected for revascularization.

25 4. The method of claim 1 , 2, or 3, wherein the angiogenic growth factor is

selected from the group consisting of a vascular endothelial cell growth factor (VEGF),
an acidic fibroblast grov^ factor (aFGF), a basic fibroblast growth factor (bFGF),
epidermal growth factor, transforming grovilh factor a and p, platelet-derived endothelial
growth factor, platelet-derived growth factor, tumor necrosis factor a, a hepatocyte

30 growrth factor (j catter factor), a colony stimulating factor (CSF), macrophage-CSF(M-
CSF), granulocyte/macrophage CSF and nitric oxide synthase (NOS).

wo 00/62798 PCT/USOO/09765

-59-

5. The method of claim 1, 2, or 3, wherein administering comprises
administering a VEGF nucleic acid to the subject.

6. The method of claim 1, 2, or 3, wherein administering comprises
administering a VEGF nucleic acid and a bPGF nucleic acid to the subject.

7. The method of claim 1, 2, or 3, wherein the angiogenic growth factor is a
VEGF nucleic acid encoding a VEGF polypeptide selected from the group consisting of:

(a) VEGF A

(b) VEGFB

(e)phVEGF,2,;
(OphVEGFns;

(g) phVEGFi65; and

(h) phVEGF,89.

8. The method of claim 1, 2, or 3, wherein administering is selected from the
group consisting of intravenous administration, intra-arterial administration and
intramuscular administration.

9. The method of claim 1, 2, or 3, wherein administering is by intramuscular
15 administration.

10. The method of claim 1, 2, or 3, wherein the angiogenic growth factor is an
angiogenic growth factor nucleic acid.

1 1 . The method of claim 1, 2, or 3, wherein the angiogenic growth factor is an
angiogenic growth factor polypeptide.

20 12. The method of claim 1 1 , wherein the angiogenic growth factor is a VEGF

polypeptide.

13. The method of claim 1 1 , wherein the angiogenic growth factor is a VEGF
polypeptide selected from the group consisting of:

(a) VEGF A
25 (b) VEGF B

(d) VEGF D:

(e)phVEGF,2i;
(OphVEGFns;

(g) phVEGFi65; and

(h) phVEGF,89.

14. The method of claim 1 , 2, or 3, wherein the condition is selected from the
group consisting of:
30 (a) an ischemic peripheral neuropathy;

(b) a toxin-induced peripheral neuropathy;

wo 00/62798

PCTAJSOO/09765

-60-

(d) a genetically acquired peripheral neuropatiiy.

1 5. The method of claim 1 , 2, or 3, wherein the condition is an ischemic
peripheral neuropathy.

1 6. The method of claim 1 5, wherein the ischemic peripheral neuropathy is a
5 diabetic peripheral neuropathy.

17. The method of claim 15, wherein the ischemic peripheral neuropathy is a
peripheral vascular disease.

18. The method of claim 14, wherein the peripheral neuropathy is a
genetically acquired peripheral neuropathy.

10 19. The method of claim 1 8, wherein the genetically acquired peripheral

neuropathy is selected from the group of conditions consisting of peroneal muscular
atrophy (Charcot-Marie-Tooth Disease, types I, II, and X), hereditary amyloid
neuropathies, hereditary sensory neuropathy (type I and type II), porphyric neuropathy,
hereditary liability to pressure palsy, Fabry's disease, adrenomyeloneuropathy, Riley-Day

15 syndrome, Dejerine-Sottas neuropathy (hereditary motor-sensory neuropathy-III),

Refsum's disease, ataxia-telangiectasia, hereditary tyrosinemia, anaphalipoproteinemia,
abetalipoproteinemia, giant axonal neuropathy, metachromatic leukodystrophy, globoid
cell leukodystrophy, Friedrich's ataxia, mononeuropathy multiplex, plexopathy, and pure
motor neuropathy.

20 20. The method of claim 14, wherein the peripheral neuropathy is a toxin-

induced peripheral neuropathy.

21 . The method of claim 20, wherein the toxin-induced peripheral neuropathy
is a neurotoxicity mediated by an agent selected from the group consisting of a
therapeutic agent, an antineoplastic agent, a food contaminant, an envirormiental

25 pollutant.

22. A method for treating a subject who has sustained a peripheral nerve
injury, comprising:

administering at least one angiogenic growth factor to a subject in need of
such treatment in an amount effective to enhance peripheral nerve regeneration.
30 23. The method of claim 22, wherein the subject is otherwise free of

symptoms calling for treatment with an angiogenic growth factor.

wo 00/62798

PCT/US00/0976S

I/I

70-
60-
50-
40-

30"
20-

lOH

Q Non- diabetic
Q Diabetic

n-s.

* *

Saline Saline VEGF

SO-
TO -
60
50-
o 40-

E 30H
20-
10-

Q Non -diabetic
Q Diabetic

n.s.

* *

Saline Saline VEGF

FIG.IA

FIG. IB

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "USPTO Patents Application 09869475"

See other formats